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Purple fleshed sweet potato (Ipomoea batatas L.) is one of the natural ingredients that contain natural colour pigments are anthocyanin. Anthocyanin is a water soluble pigments which is naturally found in various type of plants. This study aims to make lipstick preparations using the purple fleshed sweet potato extract (Ipomoea batatas L.) as a natural dye. The method is used to get anthocyanin as a color of lipstick by using maceration extraction method. The weight of sample is 250 grams of dried fleshed purple sweet potato. The liquid for maceration used of 95% ethanol. The condition of maceration is acid by using 2% of citric acid. The filtrate of maceration process must be thickened. The basis of lipstick components consisted of cera alba, lanolin, cetyl alcohol, paraffin solid, oleum ricini, propylene glycol, and nipasol. The concentration of fleshed purple sweet potato extract is 0%, 2.4%, 4.5% and 14.6%. The result of research is that the lipstick is easy to apply, unstable color, homogenous, melting point above 500C, pH of 6 and all lipstick have an breaking point when load at 330 grams. Extract of purple fleshed sweet potato can’t be used as a pigment color in the manufacture of lipstick but additional material is needed that can keep the lipstick color from being degraded by pH, storage temperature, light, enzymes, oxygenation, sugar, structural differences in anthocyanins and concentrations of anthocyanins.

Gelatin is a type of protein that is extracted from collagen of skin, bone, or ligament (animal connective tissue) tissue. Cow bone, cowhide and pig skin are the materials commonly used to obtain gelatin. The purpose of this research was to determine the difference of NaOH concentration as soaking agent on gelatin making from skin of chicken leg (Gallus gallus domesticus) for 72 hours of soaking. Fresh skin of chicken leg weighing 250 g were immersed in NaOH solution with concentrations of 1%, 1.5%, and 3% for 72 hours, then extracted using n-hexane solution: alcohols in a ratio of 1: 3 and dried to obtain gelatin. The resulting gelatin is then analyzed for physicochemical characteristics such as sensory, yield, pH test, and water content test. The results of this study, mentioned that the concentration of 1% NaOH solution produces gelatin under average of yield of 6 g, neutral pH and water content of 11%. The concentration of 1.5% NaOH solution produces gelatin under average of yield 8 g, pH 7 and water content of 13%. The concentration of 3%NaOH solution produces gelatin under average of yield 4.144 g, pH 7 and water content of 7.11%. Based on the result, it can be concluded that a good solution of NaOH to get high yield under a concentration of 1.5% solution. The gelatin obtained under 1.5% solution in accordance with the standard gelatin. The characteristics of standard gelatin are colorless to yellow, odorless, tasteless, pH 7 (neutral), and moisture content below 16%.

Lipstick is one of the most used cosmetics by women. According to Tranggano and Latifah (2007), lipstick is a cosmetic applied to the lips, to protect the lips in cold and dry air so that the lips are not easy to dry and crack. Lipstick is used by women for a long time so that lipstick should be safe and do not cause irritation. Chemicals substances in the manufacture of lipstick can accidentally be consumed and long-term use so that its accumulation in the body would be harmful to health. Synthetic lipstick color is one part of the dangerous lipstick making material. Synthetic substances can be replaced with natural dyes. The use of natural dye limits on cosmetics is 5-15% (Mercado, 1991). One example of natural dyes that can be used is the dye from the Jati leaves (Tectona grandis L.f). This study aim is to prove that Jati leaves (Tectona grandis L.f) can be used as a natural dye in the material of lipstick. The method used in this research is the method of extraction maceration with jati leaf samples of 300 grams and ethanol of 96%. Component of lipstick formula which are cera alba, lanolin, vaseline, cetyl alcohol, paraffin solid, oleum ricini, oleum rosae, propylene glycol, butyl hydroxytoluene, methyl paraben, and the addition Jati leaf extract under the concentration of 0%, 18% , and 22%. The material of lipstick is easy to apply, stable, brown color, homogeneous, melting point of 600C, range pH between 3 at 4 and lipstick have a breaking point of 500 grams. Jati leaf extract (Tectona grandis L.f) can be used as a dye or color of lipstick.

Aquaporins (AQPs) are transmembrane channels initially discovered for their role in water flux facilitation through biological membranes. Over the years, a much more complex and subtle picture of these channels appeared, highlighting many other solutes accommodated by AQPs and a dense regulatory network finely tuning cell membranes' water permeability. At the intersection between several transduction pathways (e.g., cell volume regulation, calcium signaling, potassium cycling, etc.), this wide and ancient protein family is considered an important therapeutic target for cancer treatment and many other pathophysiologies. However, a precise and isoform-specific modulation of these channels function is still challenging. Among the modulators of AQPs functions, cations have been shown to play a significant contribution, starting with mercury being historically associated with the inhibition of AQPs since their discovery. While the comprehension of AQPs modulation by cations has improved, a unifying molecular mechanism integrating all current knowledge is still lacking. In an effort to extract general trends, we reviewed all known modulations of AQPs by cations to capture a first glimpse of this regulatory network. We paid particular attention to the associated molecular mechanisms and pinpointed the residues involved in cation binding and in conformational changes tied up to the modulation of the channel function.

Inherited cardiomyopathies are common cardiac diseases worldwide, leading in the late stage to heart failure and death. The most promising treatments against these diseases are small molecules directly modulating the force produced by β-cardiac myosin, the molecular motor driving heart contraction. Omecamtiv mecarbil and Mavacamten are two such molecules that completed phase 3 clinical trials, and the inhibitor Mavacamten is now approved by the FDA. In contrast to Mavacamten, Omecamtiv mecarbil acts as an activator of cardiac contractility. Here, we reveal by X-ray crystallography that both drugs target the same pocket and stabilize a pre-stroke structural state, with only few local differences. All-atom molecular dynamics simulations reveal how these molecules produce distinct effects in motor allostery thus impacting force production in opposite way. Altogether, our results provide the framework for rational drug development for the purpose of personalized medicine., (© 2024. The Author(s).)

In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of photoreceptors (PRs) and vision. Among those, RPE cells phagocytose under a circadian rhythm photoreceptor outer segment (POS) tips that are constantly subjected to light rays and oxidative attacks. The MerTK tyrosine kinase receptor is a key element of this phagocytic machinery required for POS internalization. Recently, we showed that MerTK is subjected to the cleavage of its extracellular domain to finely control its function. In addition, monocytes in retinal blood vessels can migrate inside the inner retina and differentiate into macrophages expressing MerTK, but their role in this context has not been studied yet. We thus investigated the ocular phenotype of MerTK cleavage-resistant (MerTK CR ) mice to understand the relevance of this characteristic on retinal homeostasis at the RPE and macrophage levels. MerTK CR retinae appear to develop and function normally, as observed in retinal sections, by electroretinogram recordings and optokinetic behavioral tests. Monitoring of MerTK CR and control mice between the ages of 3 and 18  months showed the development of large degenerative areas in the central retina as early as 4 months when followed monthly by optical coherence tomography (OCT) plus fundus photography (FP)/autofluorescence (AF) detection but not by OCT alone. The degenerative areas were associated with AF, which seems to be due to infiltrated macrophages, as observed by OCT and histology. MerTK CR RPE primary cultures phagocytosed less POS in vitro , while in vivo , the circadian rhythm of POS phagocytosis was deregulated. Mitochondrial function and energy production were reduced in freshly dissected RPE/choroid tissues at all ages, thus showing a metabolic impairment not present in macrophages. RPE anomalies were detected by electron microscopy, including phagosomes retained in the apical area and vacuoles. Altogether, this new mouse model displays a novel phenotype that could prove useful to understanding the interplay between RPE and PRs in inflammatory retinal degenerations and highlights new roles for MerTK in the regulation of the energetic metabolism and the maintenance of the immune privilege in the retina., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Enderlin, Rieu, Réty, Vanoni, Roux, Dégardin, César, Augustin, Nous, Cai, Fontaine, Sennlaub and Nandrot.)

Aquaporins (AQPs) constitute a wide family of water channels implicated in all kind of physiological processes. Zinc is the second most abundant trace element in the human body and a few studies have highlighted regulation of AQP0 and AQP4 by zinc. In the present work, we addressed the putative regulation of AQPs by zinc cations in silico through molecular dynamics simulations of human AQP0, AQP2, AQP4, and AQP5. Our results align with other scales of study and several in vitro techniques, hence strengthening the reliability of this regulation by zinc. We also described two distinct putative molecular mechanisms associated with the increase or decrease in AQPs' water permeability after zinc binding. In association with other studies, our work will help deciphering the interaction networks existing between zinc and channel proteins.

Ménière's disease (MD) is characterized by an abnormal dilatation of the endolymphatic compartment called endolymphatic hydrops and is associated with fluctuating hearing losses and vertigo. Corticosteroid treatment is typically administered for its anti-inflammatory effects to MD patients. However, we recently described for the first time a direct interaction of two corticosteroids (dexamethasone and cortisol) with human AQP2 which strongly inhibited water fluxes. From these initial studies, we proposed an AQPs Corticosteroids Binding Site (ACBS). In the present work, we tested the interaction of 10 molecules associated to the steroid family for this putative ACBS. We observed a wide diversity of affinity and inhibitory potential of these molecules toward AQP2 and discussed the implications for inner ear physiology. Among the tested compounds, cholecalciferol, calcitriol and oestradiol were the most efficient AQP2 water permeability inhibitors., Competing Interests: The authors declare that this study received funding from the MEDEL company. The funder had the following involvement with the study: Decision to submit the article for publication. The funder was not involved in the study design, collection, analysis, interpretation of data or the writing of this article. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Mom, Réty, Mocquet and Auguin.)

Aquaporins (AQPs) constitute a wide and ancient protein family of transmembrane channels dedicated to the regulation of water exchange across biological membranes. In plants, higher numbers of AQP homologues have been conserved compared to other kingdoms of life such as in animals or in bacteria. As an illustration of this plant-specific functional diversity, plasma membrane intrinsic proteins (PIPs, i.e., a subfamily of plant AQPs) possess a long intracellular loop D, which can gate the channel by changing conformation as a function of the cellular environment. However, even though the closure of the AQP by loop D conformational changes is well described, the opening of the channel, on the other hand, is still misunderstood. Several studies have pointed to phosphorylation events as the trigger for the transition from closed- to open-channel states. Nonetheless, no clear answer has been obtained yet. Hence, in order to gain a more complete grasp of plant AQP regulation through this intracellular loop D gating, we investigated the opening of the channel in silico through molecular dynamics simulations of the crystallographic structure of Spinacia oleracea PIP2;1 ( So PIP2;1). Through this technique, we addressed the mechanistic details of these conformational changes, which eventually allowed us to propose a molecular mechanism for PIP functional regulation by loop D phosphorylation. More precisely, our results highlight the phosphorylation of loop D serine 188 as a trigger of So PIP2;1 water channel opening. Finally, we discuss the significance of this result for the study of plant AQP functional diversity.

Pif1 proteins are DNA helicases belonging to Superfamily 1, with 5' to 3' directionality. They are conserved from bacteria to human and have been shown to be particularly important in eukaryotes for replication and nuclear and mitochondrial genome stability. However, Pif1 functions in bacteria are less known. While most Pif1 from mesophilic bacteria consist of the helicase core with limited N-terminal and C-terminal extensions, some Pif1 from thermophilic bacteria exhibit a C-terminal WYL domain. We solved the crystal structures of Pif1 helicase cores from thermophilic bacteria Deferribacter desulfuricans and Sulfurihydrogenibium sp. in apo and nucleotide bound form. We show that the N-terminal part is important for ligand binding. The full-length Pif1 helicase was predicted based on the Alphafold algorithm and the nucleic acid binding on the Pif1 helicase core and the WYL domain was modelled based on known crystallographic structures. The model predicts that amino acids in the domains 1A, WYL, and linker between the Helicase core and WYL are important for nucleic acid binding. Therefore, the N-terminal and C-terminal extensions may be necessary to strengthen the binding of nucleic acid on these Pif1 helicases. This may be an adaptation to thermophilic conditions.

Aquaporins (AQPs) are water channels widely distributed in living organisms and involved in many pathophysiologies as well as in cell volume regulations (CVR). In the present study, based on the structural homology existing between mineralocorticoid receptors (MRs), glucocorticoid receptors (GRs), cholesterol consensus motif (CCM) and the extra-cellular vestibules of AQPs, we investigated the binding of corticosteroids on the AQP family through in silico molecular dynamics simulations of AQP2 interactions with cortisol. We propose, for the first time, a putative AQPs corticosteroid binding site (ACBS) and discussed its conservation through structural alignment. Corticosteroids can mediate non-genomic effects; nonetheless, the transduction pathways involved are still misunderstood. Moreover, a growing body of evidence is pointing toward the existence of a novel membrane receptor mediating part of these rapid corticosteroids' effects. Our results suggest that the naturally produced glucocorticoid cortisol inhibits channel water permeability. Based on these results, we propose a detailed description of a putative underlying molecular mechanism. In this process, we also bring new insights on the regulatory function of AQPs extra-cellular loops and on the role of ions in tuning the water permeability. Altogether, this work brings new insights into the non-genomic effects of corticosteroids through the proposition of AQPs as the membrane receptor of this family of regulatory molecules. This original result is the starting point for future investigations to define more in-depth and in vivo the validity of this functional model.

Ménière's disease is a chronic illness characterized by intermittent episodes of vertigo associated with fluctuating sensorineural hearing loss, tinnitus and aural pressure. This pathology strongly correlates with a dilatation of the fluid compartment of the endolymph, so-called hydrops. Dexamethasone is one of the therapeutic approaches recommended when conventional antivertigo treatments have failed. Several mechanisms of actions have been hypothesized for the mode of action of dexamethasone, such as the anti-inflammatory effect or as a regulator of inner ear water homeostasis. However, none of them have been experimentally confirmed so far. Aquaporins (AQPs) are transmembrane water channels and are hence central in the regulation of transcellular water fluxes. In the present study, we investigated the hypothesis that dexamethasone could impact water fluxes in the inner ear by targeting AQP2. We addressed this question through molecular dynamics simulations approaches and managed to demonstrate a direct interaction between AQP2 and dexamethasone and its significant impact on the channel water permeability. Through compartmentalization of sodium and potassium ions, a significant effect of Na+ upon AQP2 water permeability was highlighted as well. The molecular mechanisms involved in dexamethasone binding and in its regulatory action upon AQP2 function are described.

Aquaporins are transmembrane water channels found in almost every living organism. Numerous studies have brought a good understanding of both water transport through their pores and the regulations taking place at the molecular level, but subtleties remain to be clarified. Recently, a voltage-related gating mechanism involving the conserved arginine of the channel's main constriction was captured for human aquaporins through molecular dynamics studies. With a similar approach, we show that this voltage-gating could be conserved among this family and that the underlying mechanism could explain part of plant AQPs diversity when contextualized to high ionic concentrations provoked by drought. Finally, we identified residues as adaptive traits which constitute good targets for drought resistance plant breeding research., (© 2020 Federation of European Biochemical Societies.)

Roses use a non-canonical pathway involving a Nudix hydrolase, RhNUDX1, to synthesize their monoterpenes, especially geraniol. Here we report the characterization of another expressed NUDX1 gene from the rose cultivar Rosa x wichurana, RwNUDX1-2. In order to study the function of the RwNUDX1-2 protein, we analyzed the volatile profiles of an F 1 progeny generated by crossing R. chinensis cv. 'Old Blush' with R. x wichurana. A correlation test of the volatilomes with gene expression data revealed that RwNUDX1-2 is involved in the biosynthesis of a group of sesquiterpenoids, especially E,E-farnesol, in addition to other sesquiterpenes. In vitro enzyme assays and heterologous in planta functional characterization of the RwNUDX1-2 gene corroborated this result. A quantitative trait locus (QTL) analysis was performed using the data of E,E-farnesol contents in the progeny and a genetic map was constructed based on gene markers. The RwNUDX1-2 gene co-localized with the QTL for E,E-farnesol content, thereby confirming its function in sesquiterpenoid biosynthesis in R. x wichurana. Finally, in order to understand the structural bases for the substrate specificity of rose NUDX proteins, the RhNUDX1 protein was crystallized, and its structure was refined to 1.7 Å. By molecular modeling of different rose NUDX1 protein complexes with their respective substrates, a structural basis for substrate discrimination by rose NUDX1 proteins is proposed., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.

The DEAH box helicase Prp43 is a bifunctional enzyme from the DEAH/RHA helicase family required both for the maturation of ribosomes and for lariat intron release during splicing. It interacts with G-patch domain containing proteins which activate the enzymatic activity of Prp43 in vitro by an unknown mechanism. In this work, we show that the activation by G-patch domains is linked to the unique nucleotide binding mode of this helicase family. The base of the ATP molecule is stacked between two residues, R159 of the RecA1 domain (R-motif) and F357 of the RecA2 domain (F-motif). Using Prp43 F357A mutants or pyrimidine nucleotides, we show that the lack of stacking of the nucleotide base to the F-motif decouples the NTPase and helicase activities of Prp43. In contrast the R159A mutant (R-motif) showed reduced ATPase and helicase activities. We show that the Prp43 R-motif mutant induces the same phenotype as the absence of the G-patch protein Gno1, strongly suggesting that the processing defects observed in the absence of Gno1 result from a failure to activate the Prp43 helicase. Overall we propose that the stacking between the R- and F-motifs and the nucleotide base is important for the activity and regulation of this helicase family.

Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins.

In eukaryotes, three of the four ribosomal RNAs (rRNAs)—the 5.8S, 18S, and 25S/28S rRNAs—are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit., (© 2015 Madru et al.; Published by Cold Spring Harbor Laboratory Press.)

RNA helicases from the DEAH/RHA family are present in all the processes of RNA metabolism. The function of two helicases from this family, Prp2 and Prp43, is regulated by protein partners containing a G-patch domain. The G-patch is a glycine-rich domain discovered by sequence alignment, involved in protein-protein and protein-nucleic acid interaction. Although it has been shown to stimulate the helicase's enzymatic activities, the precise role of the G-patch domain remains unclear. The role of G-patch proteins in the regulation of Prp43 activity has been studied in the two biological processes in which it is involved: splicing and ribosome biogenesis. Depending on the pathway, the activity of Prp43 is modulated by different G-patch proteins. A particular feature of the structure of DEAH/RHA helicases revealed by the Prp43 structure is the OB-fold domain in C-terminal part. The OB-fold has been shown to be a platform responsible for the interaction with G-patch proteins and RNA. Though there is still no structural data on the G-patch domain, in the current model, the interaction between the helicase, the G-patch protein, and RNA leads to a cooperative binding of RNA and conformational changes of the helicase.

During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53-MDM2 pathway. This work presents the functional and structural characterization of the Fap7-Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation., Competing Interests: The authors have declared that no competing interests exist.

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.

We report a functional type I toxin-antitoxin (TA) module expressed by a human pathogen, Staphylococcus aureus. TA systems consist of stable toxins and labile antitoxins encoded within small genetic modules widespread in eubacteria and archaea. TA genes provide stress adaptation and protection against DNA loss or invasion. The genes encoding the SprA1 toxic peptide (PepA1) and the SprA1(AS) RNA antitoxin are within a pathogenicity island on opposite strands and possess a 3' overlap. To prevent peptide toxicity during S. aureus growth, PepA1 expression from stable (half-life > 3 h) SprA1 is repressed by elevated amounts of unstable (half-life = ∼10 mn) SprA1(AS). In vivo, PepA1 localizes at the bacterial membrane and triggers S. aureus death. Based on NMR and CD data, its solution structure was solved and is a long bent, interrupted helix. Molecular dynamics simulations indicate that PepA1 compaction and helical content fluctuate in accordance with its cytoplasm or membrane location. When inserted into the S. aureus membrane, the PepA1 conformation switches to a ∼7-nm-long continuous helix, presumably forming pores to alter membrane integrity. PepA1 expression is induced upon acidic and oxidative stresses by reducing SprA1(AS) levels. As an altruistic behavior during infection, some cells may induce the expression of that toxin that would facilitate departure from the host immune cells for spreading.

Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.

FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in Pseudomonas aeruginosa. We present here two crystallographic structures of the EAL domain of FimX (residues 438-686): one of the apo form and the other of a complex with 5'-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5'-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains.

SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins (RNPs) required for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. SHQ1 binds dyskerin/NAP57, the catalytic subunit of human H/ACA RNPs, and this interaction is modulated by mutations causing X-linked dyskeratosis congenita. We report the crystal structure of the C-terminal domain of yeast SHQ1, Shq1p, and its complex with yeast dyskerin/NAP57, Cbf5p, lacking its catalytic domain. The C-terminal domain of Shq1p interacts with the RNA-binding domain of Cbf5p and, through structural mimicry, uses the RNA-protein-binding sites to achieve a specific protein-protein interface. We propose that Shq1p operates as a Cbf5p chaperone during RNP assembly by acting as an RNA placeholder, thereby preventing Cbf5p from nonspecific RNA binding before association with an H/ACA RNA and the other core RNP proteins.

Retroviral integrases are vital enzymes in the viral life cycle and thus are important targets for antiretroviral drugs. The structure of the catalytic core domain of the integrase from human foamy virus, which is related to HIV-1, has been solved. The structure of the protein is presented in two different crystal forms, each containing several molecules in the asymmetric unit, with and without the essential manganese or magnesium ion, and the structures are compared in detail. This allows regions of high structural variability to be pinpointed, as well as the effect of divalent cations on the conformation of the catalytic site.

The phenolic methyl ether 3,5-dimethoxytoluene (DMT) is a major scent compound of many modern rose varieties, and its fragrance participates in the characteristic "tea scent" that gave their name to Tea and Hybrid Tea roses. Among wild roses, phenolic methyl ether (PME) biosynthesis is restricted to Chinese rose species, but the progenitors of modern roses included both European and Chinese species (e.g., Rosa chinensis cv Old Blush), so this trait was transmitted to their hybrid progeny. The last steps of the biosynthetic pathways leading to DMT involve two methylation reactions catalyzed by the highly similar orcinol O-methyltransferases (OOMT) 1 and 2. OOMT1 and OOMT2 enzymes exhibit different substrate specificities that are consistent with their operating sequentially in DMT biosynthesis. Here, we show that these different substrate specificities are mostly due to a single amino acid polymorphism in the phenolic substrate binding site of OOMTs. An analysis of the OOMT gene family in 18 species representing the diversity of the genus Rosa indicated that only Chinese roses possess both the OOMT2 and the OOMT1 genes. In addition, we provide evidence that the Chinese-rose-specific OOMT1 genes most probably evolved from an OOMT2-like gene that has homologues in the genomes of all extant roses. We propose that the emergence of the OOMT1 gene may have been a critical step in the evolution of scent production in Chinese roses.

The lethal disease anthrax is propagated by spores of Bacillus anthracis, which can penetrate into the mammalian host by inhalation, causing a rapid progression of the disease and a mostly fatal outcome. We have solved the three-dimensional structure of the major surface protein BclA on B. anthracis spores. Surprisingly, the structure resembles C1q, the first component of complement, despite there being no sequence homology. Although most assays for C1q-like activity, including binding to C1q receptors, suggest that BclA does not mimic C1q, we show that BclA, as well as C1q, interacts with components of the lung alveolar surfactant layer. Thus, to better recognize and invade its hosts, this pathogenic soil bacterium may have evolved a surface protein whose structure is strikingly close to a mammalian protein.

The structure determination of the recombinant form of BclA, the major protein component of Bacillus anthracis exosporium, involved soaking in a high concentration of potassium iodide as the means of obtaining a good-quality heavy-atom derivative. The data to 2 angstroms resolution collected on a laboratory source were of sufficient quality to allow successful phasing and chain tracing by automated methods.

Annexin A8 is a relatively infrequent and poorly studied member of this large family of calcium-binding and membrane-binding proteins. It is, however, associated with a specific disease, acute promyelocytic leukemia. We have solved its three-dimensional structure, which includes a moderately long and intact N terminus. The structure is closest to that of annexin A3 and highlights several important regions of inherent flexibility in the annexin molecule. The N terminus resembles that of annexin A3, as it lies along the concave surface of the molecule and inserts partially into the hydrophilic channel in its centre. Since both annexins A3 and A8 are expressed in promyelocytic cells during their differentiation, the similarity in their structures might suggest a functional relationship.

The mammalian Int-6 protein has been characterized as a subunit of the eIF3 translation initiation factor and also as a transforming protein when its C-terminal part is deleted. It includes a protein domain, which also exists in various subunits of eIF3, of the 26S proteasome and of the COP9 signalosome (CSN). By performing a two-hybrid screen with Int-6 as bait, we have isolated subunits belonging to all three complexes, namely eIF3-p110, Rpt4, CSN3 and CSN6. The results of transient expression experiments in COS7 cells confirmed the interaction of Int-6 with Rpt4, CSN3 and CSN6, but also showed that Int-6 is able to bind another subunit of the CSN: CSN7a. Immunoprecipitation experiments performed with the endogenous proteins showed that Int-6 binds the entire CSN, but in low amount, and also that Int-6 is associated with the 26S proteasome. Taken together these results show that the Int-6 protein can bind the three complexes with various efficiencies, possibly exerting a regulatory activity in both protein translation and degradation.

The Int-6 protein has been shown to be a subunit of eukaryotic translation initiation factor 3 (eIF3) and to play a role in the control of cell growth. By immunoprecipitation experiments and mass spectrometry analyses, we identified a human protein previously known as HSPC021 that is associated with Int-6. Exposure of Jurkat cells to the phosphatase inhibitor H(2)O(2) triggers a marked phosphorylation on tyrosine of HSPC021. Several experiments were performed to evaluate whether this protein is associated with eIF3. It was observed that HSPC021 coelutes with Int-6 and eIF3 in gel filtration, coimmunoprecipitates with eIF3, and is incorporated into eIF3 both in rabbit reticulocyte lysates and in COS7 cells. A direct protein-protein interaction occurs between HSPC021 and Int-6, but the analysis of different mutants of HSPC021 indicated that a larger region of the protein is necessary for incorporation into eIF3 as compared with binding to Int-6. Taken together, our results establish that HSPC021 is tightly associated with the mammalian translation initiation factor eIF3. Analysis of the primary sequence of HSPC021 from different species revealed the presence of a tetratricopeptide repeat, a proteasome-COP9 (constitutive photomorphogenesis 9) signalosome-initiation factor 3 domain along with a Pumilio FBF repeat. These protein motifs are also present in subunits of eIF3, of the lid of the 26 S proteasome, and of the COP9 signalosome.

The (Anx2)(2)(p11)(2) heterotetramer has been implicated in endo- and exocytosis in vivo and in liposome aggregation in vitro. Here we report on the modelling of the heterotetramer complex using docking algorithms. Two types of models are generated-heterotetramer and heterooctamer. On the basis of the agreement between the calculated (X-ray) electron density and the observed projected density from cryo-electron micrographs on the one hand, and calculated energy criteria on the other hand, the heterotetramer models are proposed as the most probable, and one of them is selected as the best model. Analysis of this model at an atomic level suggests that the interaction between the Anx2 core and p11 has an electrostatic character, being stabilised primarily through charged residues.

The EF-hand motif is the most common calcium-binding motif found in proteins. Several high-resolution structures containing different metal ions bound to EF-hand sites have given new insight into the modulation of their binding affinities. Recently determined structures of members of several newly identified protein families that contain the EF-hand motif in some of their domains, as well as of their complexes with target molecules, are throwing light on the surprising variety of functions that can be served by this simple and ingenious structural motif.

Background: S100C (S100A11) is a member of the S100 calcium-binding protein family, the function of which is not yet entirely clear, but may include cytoskeleton assembly and dynamics. S100 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop. Like several other members of the family, S100C forms a homodimer. A number of S100 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids. Structural studies have been undertaken to understand the basis of these interactions., Results: We have solved the crystal structure of a complex of calcium-loaded S100C with a synthetic peptide that corresponds to the first 14 residues of the annexin I N terminus at 2.3 A resolution. We find a stoichiometry of one peptide per S100C monomer, the entire complex structure consisting of two peptides per S100C dimer. Each peptide, however, interacts with both monomers of the S100C dimer. The two S100C molecules of the dimer are linked by a disulphide bridge. The structure is surprisingly close to that of the p11-annexin II N-terminal peptide complex solved previously. We have performed competition experiments to try to understand the specificity of the S100-annexin interaction., Conclusions: By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.

Several annexins have been shown to bind proteins that belong to the S100 calcium-binding protein family. The two best-characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N-terminus and of S100C with annexin I N-terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42-55), we propose a computer generated model for the entire annexin II/p11 complex., (Copyright 2000 Academic Press.)

The aggregation and membrane fusion properties of annexin II are modulated by the association with a regulatory light chain called p11.p11 is a member of the S100 EF-hand protein family, which is unique in having lost its calcium-binding properties. We report the first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone. The basic unit for p11 is a tight, non-covalent dimer. In the complex, each annexin II peptide forms hydrophobic interactions with both p11 monomers, thus providing a structural basis for high affinity interactions between an S100 protein and its target sequence. Finally, p11 forms a disulfide-linked tetramer in both types of crystals thus suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.

The Src homologous and collagen-like (SHC) protein plays an essential role in signal transduction pathways in that it participates in the chain of events that leads to the activation of the protein Ras. The crystal structure of the SH2 domain of SHC has been determined using the method of multiple isomorphous replacement at a resolution of 2.5 A. The SH2 domain of SHC is similar in fold to other SH2 domains. The peptide-binding surfaces resemble that of the SH2 domain of Src in that a deep pocket is formed where the third amino acid C-terminal to the phosphotyrosine can insert. A novel feature of this structure is the observation of a disulfide bond and an extensive dimer interface between two symmetry-related molecules. Solution studies under reducing conditions using analytical centrifugation and PAGE suggest that the SH2 domain of SHC dimerizes in a pH-dependent manner where low pH conditions (approximately 4.5) are conducive to dimer formation. Dimerization of SHC may have important biological implications in that it may promote the assembly of large heteromultimeric signaling complexes.