A novel mechanism for the modulation of the Ras-effector interaction by small molecules

When proteins require different conformations for their biological function, all these functional states have to coexist simultaneously in solution. However, the corresponding Gibbs free energy differences are usually rather high and thus the conformation with lowest energy predominates in solution whereas the populations of the states with higher energy (excited states) are very small. A stabilization of these excited states can be used as a novel principle to influence the activity of proteins by small molecules. For a proof of this principle, we selected the Ras protein that was shown by (31)P NMR spectroscopy to exist in solution in at least two different conformational states in its GTP form. One of these states shows a drastically reduced affinity to effectors. With Zn(2+)-cyclen we found a small molecule which selectively stabilizes the weak-binding state. It may serve as lead compound for the development of a new type of Ras-inhibitors.     

A novel type of allosteric regulation: functional cooperativity in monomeric proteins

Cooperative functional properties and allosteric regulation in cytochromes P450 play an important role in xenobiotic metabolism and define one of the main mechanisms of drug-drug interactions. Recent experimental results suggest that ability to bind simultaneously two or more small organic molecules can be the essential feature of cytochrome P450 fold, and often results in rich and complex pattern of allosteric behavior. Manifestations of non-Michaelis kinetics include homotropic and heterotropic activation and inhibition effects depending on the stoichiometric ratios of substrate and effector, changes in the regio- and stereospecificity of catalytic transformations, and often give rise to the clinically important drug-drug interactions. In addition, functional response of P450 systems is modulated by the presence of specific and non-specific effector molecules, metal ions, membrane incorporation, formation of homo- and hetero-oligomers, and interactions with the protein redox partners. In this article we briefly overview the main factors contributing to the allosteric effects in cytochromes P450 with the main focus on the sources of cooperative behavior in xenobiotic metabolizing monomeric heme enzymes with their conformational flexibility and extremely broad substrate specificity. The novel mechanism of functional cooperativity in P450 enzymes does not require substantial binding cooperativity, rather it implies the presence of one or more binding sites with higher affinity than the single catalytically active site in the vicinity of the heme iron.     

The topography of functional interhemispheric asymmetry in the visual cortex]

Functional interhemispheric asymmetry was investigated by evoked potentials method in experiments on ten cats under ethaminal anaesthesia at 200 points of the visual cortex during the action of binocular and monocular photic flashes of submaximal intensity. Topographic maps have been plotted of the functional interhemispheric asymmetry. In most of the animals a hemisphere dominant and non-dominant at the given moment can be singled out. Section of the callosal body leads to reduction of the functional interhemispheric asymmetry due to a decrease of the focus of maximum activity in the dominant hemisphere and its increase in the non-dominant one. A mozaic pattern of functional interhemispheric asymmetry has been demonstrated, as expressed in the existence of zones of inverse dominance along with prevailing zones of direct dominance. Section of the callosal body produced a decrease in the area of direct dominance and an increase in that of inverse dominance. Absolute interhemispheric asymmetry was most pronounced in the central part of the visual cortex (field 18 and its medial boundary) and the relative one, on the periphery of the visual area (fields 17 and 19).     

A conserved interdomain interaction is a determinant of folding cooperativity in the GST fold

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.     

Extracallosal channels of interhemispheric transmission of visual information

Evoked potentials (EP) in response to light flashes were recorded in cats with different degree of optical tract disconnection. In intact and operated on animals, the latent period of the first component of these EP was the same as in the visual cortex. The different degree of disconnection of the classic and commissural optical tract brought about an increase in the amplitude of commissural potentials. The data obtained point to the existence of the effective callosal and extracallosal volleys of interhemispheric transmission of visual information, which are also likely to pay an essential part in compensatory processes of the optical system.     

A dialogue between glia and neurons in the retina: modulation of neuronal excitability

Bidirectional signaling between neurons and glial cells has been demonstrated in brain slices and is believed to mediate glial modulation of synaptic transmission in the CNS. Our laboratory has characterized similar neuron-glia signaling in the mammalian retina. We find that light-evoked neuronal activity elicits Ca(2+) increases in Müller cells, which are specialized retinal glial cells. Neuron to glia signaling is likely mediated by the release of ATP from neurons and is potentiated by adenosine. Glia to neuron signaling has also been observed and is mediated by several mechanisms. Stimulation of glial cells can result in either facilitation or depression of synaptic transmission. Release of D-serine from Müller cells might also potentiate NMDA receptor transmission. Müller cells directly inhibit ganglion cells by releasing ATP, which, following hydrolysis to adenosine, activates neuronal A(1) receptors. The existence of bidirectional signaling mechanisms indicates that glial cells participate in information processing in the retina.     

Role of a goal in determining neuronal activity of the motor and visual areas of the rabbit cortex

In experiments on alert rabbits neuronal activity of the motor and visual cortical areas was studied in behavioural acts (BA) of grasping of a piece of plastics (P) and carrot (C) from consequently presented cups of the feeder; the animal had an opportunity to seize a C piece only after grasping and taking away from the previous cup the P piece. "Visual environment" in which BA were realized were identical; P and C pieces were identical in form and visual characteristics. Records were made of unit activity (201 cells), animal's movements (photoelectric method), EMG of the m. masseter; in parallel videorecords of behaviour were carried out. BA of P and C pieces grasping were identical in electromyo- and actographic characteristics; motor composition of these BA did not differ. 61 neurones were activated in both BA, 5--only in BA of P piece grasping, 22--only in BA of C piece grasping, i. e. 30% of neurones were activated only in one of the compared BA. Characteristics of activations, appearing in both acts could be significantly different: different frequency, connection with different stages of the compared BA. The obtained data are determined by changes of the motor and receptive fields of neurones in one BA in comparison to another, and are considered as an evidence supporting the suggestion that appearance of cortical neurones' activations in behaviour depends on BA goal and is not strictly determined by the parameters of movements and environment.     

A novel pharmatope tag inserted into the beta4 subunit confers allosteric modulation to neuronal nicotinic receptors

alpha-Bungarotoxin, the classic nicotinic antagonist, has high specificity for muscle type alpha1 subunits in nicotinic acetylcholine receptors. In this study, we show that an 11-amino-acid pharmatope sequence, containing residues important for alpha-bungarotoxin binding to alpha1, confers functional alpha-bungarotoxin sensitivity when strategically placed into a neuronal non-alpha subunit, normally insensitive to this toxin. Remarkably, the mechanism of toxin inhibition is allosteric, not competitive as with neuromuscular nicotinic receptors. Our findings argue that alpha-bungarotoxin binding to the pharmatope, inserted at a subunit-subunit interface diametrically distinct from the agonist binding site, interferes with subunit interface movements critical for receptor activation. Our results, taken together with the structural similarities between nicotinic and GABAA receptors, suggest that this allosteric mechanism is conserved in the Cys-loop ion channel family. Furthermore, as a general strategy, the engineering of allosteric inhibitory sites through pharmatope tagging offers a powerful new tool for the study of membrane proteins.     

Fucntional specialization and binocular interaction in the visual areas of rhesus monkey prestriate cortex.

If is is believed that neural mechanisms mediating stereoscopic vision may be localized in specific areas of the visual cortex, then it becomes necessary to be able to define these areas adequately. This is no easy matter in the rhesus monkey, an animal close to man, where the cytoarchitecturally uniform prestriate cortex is folded into deep sulci with secondary gyri. One way around this awkward problem is to use the callosal connections of the prestriate cortex as the anatomical landmarks. Callosal connections are restricted to regions at which the vertical meridian is represented. Since the visual fields, including the vertical meridian, are separately represented in each area, each has its own callosal connections. These are of great help in defining some of the boundaries of these areas, since the boundaries often coincide with the representation of the vertical meridian. With the visual areas thus defined anatomically, it becomes relatively easy to assign recordings to particular areas. Studies of binocular interactions in these areas reveal that most cells in all prestriate areas are binocularly driven. Hence, theoretically, all of the prestriate areas are candidates for stereoscopic mechanisms. The degree of binocular interaction varies from cell to cell. At the two extremes are cells which either respond to monocular stimulation only and are inhibited by binocular stimulation or ones which respond to binocular stimulation only. Changing, as opposed to fixed, disparity is signalled by two types of cells. In one category are cells activated in opposite directions for the two eyes. Such cells are always binocularly driven. In the other category are cells, some of which are monocularly activated, that are capable of responding to changing image size. In the monkey, both these categories of cells have so far been found in the motion area of the superior temporal sulcus only.     

Types of correlative relations of the neuronal impulses of the visual and sensomotor areas of the neocortex in the rabbit

Neuronal activity of the visual and sensorimotor cortical areas was simultaneously recorded in intersignal intervals in rabbits with conditioned reflexes to light and sound. ES-1020 computer built cross-correlation (CCH) and autocorrelation (ACH) histograms of impulse activity of the se neurones. Analysis of CCH form, built with a bin of 2 ms gave no convincing proofs of the presence of synaptic connections between neurone of the visual and sensorimotor cortical areas and of influences from a common source with few switchings on interneurones. Analysis of CCH built with bins of 10 and 30 ms, allowed to single out primary and secondary peaks and troughs. Wide primary peaks with the base covering the beginning of the coordinates, were met in 51.9% of cases; wide peaks, greatly shifted relatively to the coordinates beginning,--in 40.9% and trough--in 7.2% of cases. Secondary peaks did not always reproduce the ACH form of impulses trains of recorded neurones. The CCH analysis allowed to suggest two basic mechanisms, eliciting the correlated discharges--action of common input and influence of the neurones of one area on the other through a number of interneurones. It was not always possible to separate these two cases by means of CCH. In a number of cases, a more complicated character of neurones interaction could be suggested.