Use of a Reverse Micelle System for Study of Oligomeric Structure of NAD<Superscript>+</Superscript>-Reducing Hydrogenase from <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> H16

Inclusion of an oligomeric enzyme, NAD⁺-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. It was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, HoxHY, is a minimal structural unit catalyzing hydrogenase reaction with an artificial electron donor, reduced methyl viologen; 3) all structural fragments containing FMN and NAD⁺/NADH-binding sites exhibit catalytic activity in diaphorase reactions with one- and two-electron acceptors; 4) small subunits, HoxY and HoxU also exhibit activity in diaphorase reactions with artificial acceptors. These results can be considered as indirect evidence that the second FMN molecule may be associated with one of the small subunits (HoxY or HoxU) of the hydrogenase from R. eutropha.__________Translated from Biokhimiya, Vol. 70, No. 6, 2005, pp. 782–789.Original Russian Text Copyright © 2005 by Tikhonova, Kurkin, Klyachko, Popov.     

Genetically programmed cell death (apoptosis)

Extensively and successfully studied problems of programmed cell death are considered. Recent evidence on apoptosis genes is presented, including the bcl-2 family and other genes with similar functions. A scheme of pathways of the main apoptosis mechanism is constructed. Examples of associations of apoptosis and diseases are presented in a special section.     

Plasmodium ookinete-secreted proteins secreted through a common micronemal pathway are targets of blocking malaria transmission

The mosquito midgut ookinete stage of the malaria parasite, Plasmodium, possesses microneme secretory organelles that mediate locomotion and midgut wall egress to establish sporogonic stages and subsequent transmission. The purpose of this study was 2-fold: 1) to determine whether there exists a single micronemal population with respect to soluble and membrane-associated secreted proteins; and 2) to evaluate the ookinete micronemal proteins chitinase (PgCHT1), circumsporozoite and TRAP-related protein (CTRP), and von Willebrand factor A domain-related protein (WARP) as immunological targets eliciting sera-blocking malaria parasite infectivity to mosquitoes. Indirect immunofluorescence localization studies in Plasmodium gallinaceum using specific antisera showed that all three proteins are distributed intracellularly with a similar granular cytoplasmic appearance and with focal concentration of PgCHT1 and PgCTRP, but not PgWARP, at the ookinete apical end. Immunogold double-labeling electron microscopy, using antisera against the membrane-associated protein CTRP and the soluble WARP, showed that these two proteins co-localized to the same micronemal population. Within the microneme CTRP was associated peripherally at the microneme membrane, whereas PgCHT1 and WARP were diffuse within the micronemal lumen. Sera produced against Plasmodium falciparum WARP significantly reduced the infectivity of P. gallinaceum to Aedes aegypti and P. falciparum to Anopheles mosquitoes. Antisera against PgCTRP and PgCHT1 also significantly reduced the infectivity of P. gallinaceum for A. aegypti. These results support the concept that ookinete micronemal proteins may constitute a general class of malaria transmission-blocking vaccine candidates.     

Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z_WT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z_WT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.Human immunodeficiency virus type 1 (HIV-1) and other retroviruses hijack the cellular Endosomal Sorting Complex Required for Transport (ESCRT) pathway to promote the detachment of virions from the cell surface and from each other (3, 21, 42, 44, 47). The ESCRT pathway was initially identified based on its requirement for the sorting of ubiquitinated cargo into multivesicular bodies (MVB) (50, 51). During MVB biogenesis, the ESCRT pathway drives the membrane deformation and fission events required for the inward vesiculation of the limiting membrane of this organelle (26, 29, 50, 51). More recently, it emerged that the ESCRT pathway is also essential for the normal abscission of daughter cells during the final stage of cell division (10, 43). Most of the components of the ESCRT pathway are involved in the formation of four heteromeric protein complexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Additional components include ALIX, which interacts both with ESCRT-I and ESCRT-III, and the AAA ATPase Vps4, which mediates the disassembly of ESCRT-III (29, 42).The deformation and scission of endocytic membranes is thought to be mediated by ESCRT-III, which, together with Vps4, constitutes the most conserved element of the pathway (23, 26, 42). Indeed, it was recently shown that purified yeast ESCRT-III induces membrane deformation (52), and in another study three subunits of yeast ESCRT-III were sufficient to promote the formation of intralumenal vesicles in an in vitro assay (61). In mammals, ESCRT-III is formed by the charged MVB proteins (CHMPs), which are structurally related and tightly regulated through autoinhibition (2, 33, 46, 53, 62). The removal of an inhibitory C-terminal domain induces polymerization and association with endosomal membranes and converts CHMPs into potent inhibitors of retroviral budding (34, 46, 53, 60, 62). Alternatively, CHMPs can be converted into strong inhibitors of the ESCRT pathway and of HIV-1 budding through the addition of a bulky tag such as green fluorescent protein (GFP) or red fluorescent protein (RFP) (27, 36, 39, 54). Retroviral budding in general is also strongly inhibited by catalytically inactive Vps4 (22, 41, 55), or upon Vsp4B depletion (31), confirming the crucial role of ESCRT-III.Retroviruses engage the ESCRT pathway through the activity of so-called late assembly (L) domains in Gag. In the case of HIV-1, the primary L domain maps to a conserved PTAP motif in the C-terminal p6 domain of Gag (24, 28) and interacts with the ESCRT-I component Tsg101 (15, 22, 40, 58). HIV-1 p6 also harbors an auxiliary L domain of the LYPx_nL type, which interacts with the V domain of ALIX (20, 35, 39, 54, 59, 63). Interestingly, Tsg101 binding site mutants of HIV-1 can be fully rescued through the overexpression of ALIX, and this rescue depends on the ALIX binding site in p6 (20, 56). In contrast, the overexpression of a specific splice variant of the ubiquitin ligase Nedd4-2 has been shown to rescue the release and infectivity of HIV-1 mutants lacking all known L domains in p6 (12, 57). Nedd4 family ubiquitin ligases had previously been implicated in the function of PPxY-type L domains, which also depend on an intact ESCRT pathway for function (4, 32, 38). However, HIV-1 Gag lacks PPxY motifs, and the WW domains of Nedd4-2, which mediate its interaction with PPxY motifs, are dispensable for the rescue of HIV-1 L domain mutants (57).ALIX also interacts with the nucleocapsid (NC) region of HIV-1 Gag (18, 49), which is located upstream of p6 and the p1 spacer peptide. ALIX binds HIV-1 NC via its Bro1 domain, and the capacity to interact with NC and to stimulate the release of a minimal HIV-1 Gag construct is shared among widely divergent Bro1 domain proteins (48). Based on these findings and the observation that certain mutations in NC cause a phenotype that resembles that of L domain mutants, it has been proposed that NC cooperates with p6 to recruit the machinery required for normal HIV-1 budding (18, 49).NC also plays a role in Gag polyprotein multimerization, and this function of NC depends on its RNA-binding activity (5-8). It has been proposed that the role of the NC-nucleic acid interaction during assembly is to promote the formation of Gag dimers (37), and HIV-1 assembly in the absence of NC can indeed be efficiently rescued by leucine zipper dimerization domains (65). Surprisingly, in this setting the L domains in p6 also became dispensable, since particle production remained efficient even when the entire NC-p1-p6 region of HIV-1 Gag was replaced by a leucine zipper (1, 65). These findings raised the possibility that the reliance of wild-type (WT) HIV-1 Gag on a functional ESCRT pathway is, at least in part, specified by NC-p1-p6. However, it also remained possible that the chimeric Gag constructs engaged the ESCRT pathway in an alternative manner.In the present report, we provide evidence supporting the first of those two possibilities. Particle production became independent of ESCRT when the entire NC-p1-p6 region was replaced by a leucine zipper, and reversion to ESCRT dependence was shown to occur as a result of restoration of p1-p6 but not of p6 alone. Furthermore, although the deletion of p1 alone had little effect in an authentic HIV-1 Gag context, the additional removal of a portion of NC improved particle production in the presence of an inhibitor of the ESCRT pathway. Together, these data imply that the NC-p1 region plays an important role in the ESCRT-dependence of HIV-1 particle production.     

The phenomenon of <Emphasis Type="Italic">Yersinia pestis</Emphasis> biofilm formation in the organism of fleas

The paper considers, for the first time, the formation of the extracellular matrix envelope (EME), or the biofilm, by Yersinia pestis as the basis determining the nature of interaction of the plague agent with the flea organism. The significance of the insect proventriculus in the process of biofilm formation is shown. The ultrastructure of the conglomerates of the plague microbe in the flea proventriculus and midgut was studied and the uniform mechanism of their formation was established. The role of Yersinia pestis biofilm in preservation of the plague microbe in the intestine of ectoparasites and in the soil of rodent burrows was discussed. PCR analysis confirmed the presence of the agent in plague infected corpses and flea feces stored at +8−10°C for 7 years and 9 months.     

Protein engineering of formate dehydrogenase

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in dehydrogenase-based synthesis of optically active compounds. Low operational stability and high production cost of native FDHs limit their application in commercial production of chiral compounds. The review summarizes the results on engineering of bacterial and yeast FDHs aimed at improving their chemical and thermal stability, catalytic activity, switch in coenzyme specificity from NAD+ to NADP+ and overexpression in Escherichia coli cells.     

Adaptivity of social systems: the problem for scientific research

The notion of adaptive evolution of social systems as of a real process of selection of the properties of such systems implies group selection. But strong evidences of effective group selection seem impossible, at least in vertebrates. However, understanding the origin of social systems adaptivity based on individual selection is difficult, as well, without analyzing the proximal mechanisms of the formation of such systems. I suppose that social systems change due to changes of individual features that underlie the proximal mechanisms of the system formation. These features are the characteristics of neurophysiological and hormonal regulatory mechanisms. They are strongly associated with intrinsic biochemical processes and are coded in the genome. Thus, the evolution of social systems is the evolution of their proximal mechanisms. At the same time, the specificity of neurophysiological and hormonal regulation determines not only social interactions, but also the individual behaviour of animals. The most important characteristics of life history, such as the regime of activity, foraging strategy, etc., are strongly affected by the same regulatory mechanisms. This view is useful for understanding the relations combining many features into an integrated and adaptive species-specific life form. I suppose that such forms emerged as evolutionary consequences of changes in regulatory mechanisms adaptive to specific environment. Thus, we have as substantial reasons to discuss adaptations of social systems to ecological features as to discuss ecological features adapted to particular social systems. The species-specificity of regulatory mechanisms is probably based on different kinds of evolutionary choice between the rapidity and the perfection of adaptation, between flexibility and stability, and between sensibility and resistibility. I think that this choice depends largely on the predictability of the environment. The less predictable it is, the more it increases the selective value of sensibility, flexibility, and rapidity of evolution. On the contrary, stable and predictable environment stimulates less rapid but more perfect adaptations. Such choices consolidate in the genome during evolution as specific features of neurophysiological and hormonal regulation systems. These specific features, in their turn, determine ecological, behavioural, and physiological species-specificity. From this point of view, evolutionary changes in social systems can be readily perceived as consequences of the selection of individuals, promoting optimal properties under particular conditional features of regulation systems. The boundary condition for this model is the absence of specificity of the characteristics of regulation systems to different forms of stress. This condition needs to be considered closely.