An enzyme in the test tube,and a transcription factor in the cell: Moonlighting proteins and cellular factors that affect their behavior

In the cell, expression levels, allosteric modulators, post‐translational modifications, sequestration, and other factors can affect the level of protein function. For moonlighting proteins, cellular factors like these can also affect the kind of protein function. This minireview discusses examples of moonlighting proteins that illustrate how a single protein can have different functions in different cell types, in different intracellular locations, or under varying cellular conditions. This variability in the kind of protein activity, added to the variability in the amount of protein activity, contributes to the difficulty in predicting the behavior of proteins in the cell.     

Nutrients and disturbance history in two Plantago species: maternal effects as a clue for observed dichotomy between resprouting and seeding strategies

We assessed the role of nutrients and disturbance experienced by mothers (maternal effects) in the growth of progeny in a pot experiment using two Plantago species. Photosynthetic capacity, biomass allocation and fecundity were measured. Offspring of plants grown in nutrient poor conditions produced more leaves, spikes and longer leaves and in case of P. lanceolata , they had also higher photosynthetic capacity. The progeny of P. media mothers that had resprouted after disturbance was favored in nutrient poor conditions whereas the progeny of undisturbed plants was favored in nutrient rich conditions.
This study demonstrates that maternal effects may play a role in the success of either a seeding or a resprouting strategy in environments with different nutrient availability. Moreover, we showed that alteration of photosynthetic capacity, even during adult stages, is a mechanism through which maternal plants may impact their progeny.     

Clonal growth and sexual reproduction: tradeoffs and environmental constraints

Clonal growth confers a number of benefits on plants, but involves some costs as well. We examined whether seed reproduction is reduced in clonal plants due to these costs. Further, we investigated whether this relationship differs for species with optima at stressful or low‐productivity sites, as a possible indication that clonality acts as insurance against reduced seed reproduction in such conditions. We evaluated 472 species for which seed production per unit area had been determined, and employed this information together with data on seed mass, height at maturity, clonal traits and optimum habitat conditions (using Ellenberg indicator values). There was a strong hyperbolic relationship between seed output and seed mass, with a scaling coefficient of ?1, indicative of a simple tradeoff relationship. We performed analyses both with and without taking phylogeny into account. Reproductive output (i.e. the product of seed output and seed size) of was lower in clonal than in non‐clonal plants (in both with and without phylogeny incorporated in the analyses); within non‐clonal species, it was high in annuals and monocarpic plants relative to nonclonal perennials. Reproductive output was lower in clonal plants with extensive lateral spread. This may be due to lower mortality of such plants, which should favor reduced reproductive output, but direct resource tradeoff may also be involved. Reproductive output in all clonal and non‐clonal plants increased with the nutrient status and light level of the species’ optimum, and decreased with moisture. Because the proportion of clonal plants in vegetation is known to decrease along the same gradients, we can infer that as sexual reproduction becomes increasingly difficult in terms of these characteristics, clonal plants may capitalize on their capacity to bypass it. However, the relationships with habitat parameters disappeared in the phylogenetically corrected analysis, indicating that habitat preferences and reproductive output evolved together.     

Amplification ratio control system for copy number variation genotyping

We describe a generic design for ratiometric analysis suitable for determination of copy number variation (CNV) class of a gene. Following two initial sequence-specific PCR priming cycles, both ends of both amplicons (one test and one reference) in a duplex reaction, are all primed by the same universal primer (UP). Following each amplification denaturation step, the UP target and its reverse complement (UP') in each strand form a hairpin. The bases immediately beyond the 3'-end of the UP and 5' of UP' are chosen such as not to base pair in the hairpin (otherwise priming is ablated). This hairpin creates a single constant environment for priming events and chaperones free 3'-ends of amplicon strands. The resultant 'amplification ratio control system' (ARCS) permits ratiometric representation of amplicons relative to the original template into PCR plateau phase. These advantages circumvent the need for real-time PCR for quantitation. Choice of different %(G+C) content for the target and reference amplicons allows liquid phase thermal melt discrimination and quantitation of amplicons. The design is generic, simple to set up and economical. Comparisons with real-time PCR and other techniques are made and CNV assays demonstrated for haptoglobin duplicon and 'chemokine (C-C motif) ligand 3-like 1' gene.     

Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1)

PRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix alpha5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b.     

Mapping the Binding Interface between an HIV-1 Inhibiting Intrabody and the Viral Protein Rev

HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb₁₉₀) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb₁₉₀-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb₁₉₀ and the Rev multimerization domains were evaluated in different assays measuring Nb₁₉₀-Rev interaction or viral production. Seven residues within Nb₁₉₀ and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb₁₉₀-Rev structural interface. This Nb₁₉₀-Rev interaction model can guide further studies of the Nb₁₉₀ effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb₁₉₀ epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.     

X-ray crystallographic and biochemical characterization of the inhibitory action of an imidazole-dioxolane compound on heme oxygenase

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.     

Factors associated with HIV infection in married or cohabitating couples in Kenya: results from a nationally representative study

Background

In order to inform prevention programming, we analyzed HIV discordance and concordance within couples in the Kenya AIDS Indicator Survey (KAIS) 2007.

Methods

KAIS was a nationally representative population-based sero-survey that examined demographic and behavioral indicators and serologic testing for HIV, HSV-2, syphilis, and CD4 cell counts in 15,853 consenting adults aged 15–64 years. We analyzed interview and blood testing data at the sexual partnership level from married or cohabitating couples. Multivariable regression models were used to identify factors independently associated with HIV discordant and concordant status.

Results

Of 3256 couples identified in the survey, 2748 (84.4%) had interview and blood testing data. Overall, 3.8% of couples were concordantly infected with HIV, and in 5.8% one partner was infected, translating to 338,000 discordant couples in Kenya. In 83.6% of HIV-infected Kenyans living in married or cohabitating couples neither partner knew their HIV status. Factors independently associated with HIV-discordance included young age in women (AOR 1.5, 95% CI: 1.2–1.8; p<0.0001), increasing number of lifetime sexual partners in women (AOR 1.5, 95% CI: 1.3–1.8; p<0.0001), HSV-2 infection in either or both partners (AOR 4.1, 95% CI: 2.3–7.2; p<0.0001), and lack of male circumcision (AOR 1.6, 95% CI: 1.0–2.5; p = 0.032). Independent factors for HIV-concordance included HSV-2 infection in both partners (AOR 6.5, 95% CI: 2.3–18.7; p = 0.001) and lack of male circumcision (AOR 1.8, 95% CI: 1.0–3.3; p = 0.043).

Conclusions

Couple prevention interventions should begin early in relationships and include mutual knowledge of HIV status, reduction of outside sexual partners, and promotion of male circumcision among HIV-uninfected men. Mechanisms for effective prevention or suppression of HSV-2 infection are also needed.