DNA mimicry by proteins as effective mechanism for regulation of activity of DNA-dependent enzymes

Modern concepts on mechanisms of DNA-dependent enzyme regulation involving specific DNA-mimicking proteins are considered. There are proteins that share structural resemblance with DNA duplexes. These include inhibitors of type I restriction-modification enzymes (Ocr and ArdA), inhibitors of DNA gyrase MfpA and QnrABS, etc. We describe here structural features of these proteins and mechanisms responsible for their interaction with DNA-dependent enzymes and then discuss perspectives of use of DNA-mimicking proteins in analysis of replication, repair, recombination, mechanisms underlying resistance to antibiotics, and also fields of applied biotechnology.     

How proteins search for their specific sites on DNA: the role of DNA conformation

It is known since the early days of molecular biology that proteins locate their specific targets on DNA up to two orders-of-magnitude faster than the Smoluchowski three-dimensional diffusion rate. An accepted explanation of this fact is that proteins are nonspecifically adsorbed on DNA, and sliding along DNA provides for the faster one-dimensional search. Surprisingly, the role of DNA conformation was never considered in this context. In this article, we explicitly address the relative role of three-dimensional diffusion and one-dimensional sliding along coiled or globular DNA and the possibility of correlated readsorption of desorbed proteins. We have identified a wealth of new different scaling regimes. We also found the maximal possible acceleration of the reaction due to sliding. We found that the maximum on the rate-versus-ionic strength curve is asymmetric, and that sliding can lead not only to acceleration, but also in some regimes to dramatic deceleration of the reaction.     

Mechanisms and therapeutic targets for neuropathic itch

Neuropathic pruritus conditions arise from structural and/or functional damage of the peripheral or central nervous system. Novel findings of pruritus specific mediators and pathways strengthen the specificity theory of pruritus transmission, however electrophysiological studies suggest that focal activation of nociceptors and distinct discharge patterns of primary afferents also contribute to the development of the sensation of pruritus. A complex interplay between excitatory and inhibitory interneurons at spinal level, non-neuronal cells and descending modulation from upper centers contributes to neuronal sensitization and clinically to the chronicity of pruritus, as well as accompanying phenomena such as alloknesis and hyperknesis. Several topical, systemic and non-pharmacological therapeutic approaches directed at distinct targets are currently available.     

Molecular targets in the search for endothelium-protecting compounds

Impairment of endothelial function forms basis for many cardiovascular diseases, therefore today it becomes an independent target for therapeutic action, and the search for new compounds possessing endothelium-protective properties is one of the prospective goals of the pharmacotherapy and medicinal chemistry. An efficient instrument to solve the problem is the use of methods of molecular modeling. Application of the methods is possible only if detailed information on three-dimensional structure and function of molecular targets—receptors and enzymes responsible for signal transduction both inside and outside endothelial cells—is available. In the review we collected the data on the structure and functions of various macromolecules involved in the process of regulation of vascular tone. The structure of endothelial NO-synthase (EC 1.14.13.39) (eNOS) responsible for synthesis of nitrogen oxide and involved in the process of vascular tone regulation is described. The importance of its substrate, L-arginine, from the point of view of eNOS activity regulation is emphasized; the data on structure and functions of L-arginine transport system are presented. Also, various pathways of eNOS activity regulation are described, including activation and competitive inhibition through binding of exogenous substances in its active center and inhibition through caveolin binding at eNOS oxygenase domain among them, as well as regulation by means of phosphorylation of individual eNOS amino acid residues by protein kinases and their dephosphorylation by phosphatases. The importance of membrane receptors of endotheliocytes as targets for substances possessing endothelium-protective activity is emphasized. Receptors of endothelin, thrombocyte activation factor, prostaglandins, bradykinin, histamine, serotonin, and protein kinase-activated receptors are among them. The importance of calcium and potassium ion channels in vessel cells for endothelium protection is emphasized. Finally, the macromolecules discussed in the review are considered as targets in the search for endothelium-protective therapeutic agents by the proposed approaches and methods of molecular modeling.     

Mechanisms of specific and nonspecific binding of architectural proteins in prokaryotic gene regulation

IHF and HU are small basic proteins of eubacteria that bind as homodimers to double-stranded DNA and bend the duplex to promote architectures required for gene regulation. These architectural proteins share a common alpha/beta fold but exhibit different nucleic acid binding surfaces and distinct functional roles. With respect to DNA-binding specificity, for example, IHF is sequence specific, while HU is not. We have employed Raman difference spectroscopy and gel mobility assays to characterize the molecular mechanisms underlying such differences in DNA recognition. Parallel studies of solution complexes of IHF and HU with the same DNA nonadecamer (5' --> 3' sequence: TC TAAGTAGTTGATTCATA, where the phage lambda H1 consensus sequence of IHF is underlined) show the following. (i) The structure of the targeted DNA site is altered much more dramatically by IHF than by HU binding. (ii) In the IHF complex, the structural perturbations encompass both the sugar-phosphate backbone and the bases of the consensus sequence, whereas only the DNA backbone is altered by HU binding. (iii) In the presence of excess protein, complexes of order higher than 1 dimer per duplex are detected for HU:DNA, though not for IHF:DNA. The results differentiate structural motifs of IHF:DNA and HU:DNA solution complexes, provide Raman signatures of prokaryotic sequence-specific and nonspecific recognition, and suggest that the architectural role of HU may involve the capability to recruit additional binding partners to even relatively short DNA sequences.     

Mechanisms of ligand discrimination by heme proteins

Several classes of heme proteins have been identified whose primary role is to sense and transport one of the three gaseous ligands: O(2), NO, or CO. A common feature shared by all of these proteins is the need to not only recognize its target ligand, but also discriminate against other heme ligands of similar size and shape. This review describes the mechanisms that each class of heme protein sensor and transporter utilizes to promote this discrimination. The common factors utilized in the recognition of each ligand are discussed.     

Mechanisms and targets for angiogenic therapy after stroke

Stroke remains a major health problem worldwide, and is the leading cause of serious long-term disability. Recent findings now suggest that strategies to enhance angiogenesis after focal cerebral ischemia may provide unique opportunities to improve clinical outcomes during stroke recovery. In this mini-review, we survey emerging mechanisms and potential targets for angiogenic therapies in brain after stroke. Multiple elements may be involved, including growth factors, adhesion molecules and progenitor cells. Furthermore, cross talk between angiogenesis and neurogenesis may also provide additional substrates for plasticity and remodeling in the recovering brain. A better understanding of the molecular interplay between all these complex pathways may lead to novel therapeutic avenues for tackling this difficult disease.     

Mechanisms and targets for angiogenic therapy after stroke

Stroke remains a major health problem worldwide, and is the leading cause of serious long-term disability. Recent findings now suggest that strategies to enhance angiogenesis after focal cerebral ischemia may provide unique opportunities to improve clinical outcomes during stroke recovery. In this mini-review, we survey emerging mechanisms and potential targets for angiogenic therapies in brain after stroke. Multiple elements may be involved, including growth factors, adhesion molecules and progenitor cells. Furthermore, cross talk between angiogenesis and neurogenesis may also provide additional substrates for plasticity and remodeling in the recovering brain. A better understanding of the molecular interplay between all these complex pathways may lead to novel therapeutic avenues for tackling this difficult disease.Key words: angiogenic therapy, stroke, neuroprotection, neurogenesis, angiogenesis, neurovascular unit, cerebral ischemia, stroke recovery     

Cannabinoids biology: the search for new therapeutic targets

Cannabinoids, in the form of marijuana plant extracts, have been used for thousands of years for a wide variety of medical conditions, ranging from general malaise and mood disorders to more specific ailments, such as pain, nausea, and muscle spasms. The discovery of tetrahydrocannabinol, the active principal in marijuana, and the identification and cloning of two cannabinoid receptors (i.e., CB1 and CB2) has subsequently led to biomedical appreciation for a family of endocannabinoid lipid transmitters. The biosynthesis and catabolism of the endocannabinoids and growing knowledge of their broad physiological roles are providing insight into potentially novel therapeutic targets. Compounds directed at one or more of these targets may allow for cannabinoid-based therapeutics with limited side effects and abuse liability.     

Cellular targets for transformation by the adenovirus E1A proteins

Three cellular proteins, including species of 300,000 daltons and 107,000 daltons as well as p105-RB, the product of the retinoblastoma susceptibility gene, stably interact with the adenovirus E1A proteins. To help determine the functional basis of these interactions, the regions of E1A that participate in these interactions were mapped using a series of deletion mutants. The 300,000 dalton and the 107,000 dalton proteins interacted with sequences within amino acids 1 to 76 and 121 to 127, respectively. Interaction with the third cellular protein, p105-RB, required the presence of sequences from two noncontiguous regions of the E1A polypeptide chain, amino acids 30 to 60 and 121 to 127. The regions of E1A that are required for these interactions coincided precisely with the regions of E1A that are required for its transforming function. These results suggest that the interactions with these cellular proteins are fundamental to the transforming activity of E1A.     

Host-bacterial coevolution and the search for new drug targets

Understanding the coevolution between humans and our microbial symbionts and pathogens requires complementary approaches, ranging from community analysis to in-depth analysis of individual genomes. Here we review the evidence for coevolution between symbionts and their hosts, the role of horizontal gene transfer in coevolution, and genomic and metagenomic approaches to identify drug targets. Recent studies have shown that our symbiotic microbes confer many metabolic capabilities that our mammalian genomes lack, and that targeting mechanisms of horizontal gene transfer is a promising new direction for drug discovery. Gnotobiotic ('germ-free') mice are an especially exciting new tool for unraveling the function of microbes, whether individually or in the context of complex communities.