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Molecularly imprinted polymer (MIP) beads for proto-hypericin recognition were prepared by suspension polymerization. In order to study the impact of monomers on the MIPs properties, various monomers such as acrylic acid (AA), hydroxyethyl methacrylate (HEMA), methacrylic acid (MAA) and itaconic acid (IA) in their combinations were crosslinked with ethylene glycol dimethacrylate (EDMA) in the presence of a complex phyto-extract template derived from Hypericum perforatum L. The synthesized MIPs and corresponding non-imprinted polymers NIPs were characterized by infrared spectroscopy analysis, morphology and thermogravimetric analysis. High-performance liquid chromatography combined with UV-Visible spectroscopy, used to investigate the recognition properties of the MIPs for various naphthodianthrones, pointed out that the MIP IA-AA system seemed to be the most adequate for favoring quantitative rebinding of proto-hypericin and proto-pseudohypericin against competitors with similar structures, like hypericin and pseudo-hypericin, which are usually present in high quantities in the primary Hypericum perforatum L. phyto-extracts.

New nanogels (NGs) with tailored properties were obtained using a mini-emulsion technique, from poly(ethylene glycol) diacrylate (PEGDA) self-crosslinking macromers of various molecular weight. By modifying synthesis parameters (hydrophilic-lipophilic balance, emulsifier and the ratio of organic-aqueous medium), optimum recipes of NGs were selected. Therefore, the molecular weight distribution and the functionalization degree of the PEGDA2000 macromer were assessed by Gel Permeation Chromatography (GPC) and Nuclear Magnetic Resonance (NMR), respectively. Furthermore, the PEGDA-NGs were investigated by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) for size distributions and morphology. DLS and TEM results confirm that these new PEGDA-NGs hold potential for biomedical applications.

In this study, composite hydrogels based on crosslinked polyethylene glycol diacrylate (PEGDA) and natural zeolite, with potential applications for the controlled release of phytoextracts, were synthesized by radical polymerization and characterized herein for the first time. The influence of various synthesis parameters, such as the use of distilled water or rose hip extract as reaction medium, or the percentage of the zeolite in the composite hydrogel upon the morphology, swelling kinetics and the rheological properties was investigated. By conducting polymerization of PEGDA in the rose hip extract, the crosslinking reaction yield was almost 100%, because of the chain transfer reactions of the polyphenols from the rose hip extract. Rose hip release tests were used to evaluate the release profiles of the natural bioactive substances from phytoextracts. The results showed that the hydrogel loaded with 3% zeolite displayed the lowest burst release and the slowest release rates of the extract.

Hypericum perforatum L., traditionally called St. John�s Wort has been used for decades as a phyto-therapeutic herb due to antidepressant, antimicrobial and antiseptic properties. As a result, the presented study evaluates the influence of the harvesting season for the Hypericum perforatum L. plant material, which is crucial for obtaining significant amounts of active principles.

In this study, composite hydrogels with interpenetrated polymer networks (IPNs), based on bacterial cellulose (BC) and poly(acrylic acid-co-N,N′-methylene-bis-acrylamide) (PAA), were synthesized by radical polymerization and characterized herein for the first time. Liquid fertilizer (LF) formulations, containing potassium, phosphorus, ammonium oxides and micronutrients, were encapsulated directly into the IPNs of the composite hydrogels during synthesis. Thermal analyses and scanning electron microscopy of control and composite xerogels highlighted the formation of IPNs between BC and PAA. Swelling determinations confirmed the influence of the crosslinker and of the liquid fertilizer concentration upon the density of the IPNs. Further rheology studies and release profiles indicated how the presence of BC and the increase of the crosslinking density of IPNs improved the mechanical strength and the release profile of LF for the innovative composite BC-PAA hydrogels. Results regarding the fertilizer release indicated that the presence of the BC led to a more controlled liberation of the fertilizer proving that this new formulation is potentially viable for application in agricultural practices.

The present paper was aimed at developing innovative hybrid composites by using cheap filler, like red mud (RM). The study importance refers to RM recycling, a recent trend in environmental science. Besides its causticity, large amounts of RM are produced yearly, calling for recovery measurements. Herein, poly (acrylic acid)- based inorganic-organic hybrid hydrogels (PAA hydrogels) incorporating RM were synthesized. Furthermore, the possibility of involving inorganic additives (kaolin and/or sodium silicate) together with RM was investigated. Incorporation of all three inorganic compounds (RM + Kaol + Sil) in hydrogels was also evaluated, novel hybrids being prepared. The obtained hybrids were characterized by different techniques (DR UV-VIS; DRIFT; TGA/DTG; SEM) and in terms of swelling behavior. DRIFT and DR UV-VIS confirmed the occurrence of characteristic bands of raw materials. Final properties (water uptake, thermal stability, porosity) can be controlled by modifying preparation conditions. Hydrogels yielded, by calcinations, iron- rich ceramic foams, able to promote water removal of sulfide ions from wastewaters.

This study presents the design of novel composites nanogels, based on poly(ethylene glycol) diacrylate and natural zeolite particles, that are able to act as materials with controlled drug delivery properties. Natural zeolite–nanogels composite, with varying zeolite contents, were obtained by an inverse mini-emulsion technique and loaded with 5-fluorouracil, a widely used chemotherapeutic drug. Herein, the possibility of adjusting final properties by means of modifying the preparation conditions was investigated. The prepared composite nanogels are characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). In light of this tunable drug-loading capability, swelling behaviour, and cytotoxicity, these composite nanogels could be highly attractive as drug reservoirs.

The synaptonemal complex (SC) keeps homologous chromosomes in close alignment during meiotic recombination. A hallmark of the SC is the presence of its constituent protein SYCP3 on the chromosome axis. During SC assembly, SYCP3 is deposited on both axes of the homologue pair, forming axial elements that fuse into the lateral element (LE) in the tripartite structure of the mature SC. We have used cryo-electron tomography and atomic force microscopy to study the mechanism of assembly and DNA binding of the SYCP3 fibre. We find that the three-dimensional architecture of the fibre is built on a highly irregular arrangement of SYCP3 molecules displaying very limited local geometry. Interaction between SYCP3 molecules is driven by the intrinsically disordered tails of the protein, with no contact between the helical cores, resulting in a flexible fibre assembly. We demonstrate that the SYCP3 fibre can engage in extensive interactions with DNA, indicative of an efficient mechanism for incorporation of DNA within the fibre. Our findings suggest that SYCP3 deposition on the chromosome axis might take place by polymerization into a fibre that is fastened to the chromosome surface via DNA binding.

The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein–protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.

In this study, TiO 2 films were successfully grown in-situ onto a FTO substrate by a hydrothermal method, using TiCl 4 as Ti precursor, and further on functionalized with a 2,4,6-trinitrotoluene-molecularly imprinted polymer (TNT-MIP) film as a preliminary step in developing a trinitrotoluene (TNT) reusable sensor to overcome the international security issues. For investigating the TiO 2 film thickness, crystalline structure and morphology, the films were autoclaved at 200 °C at different times. The X-ray diffraction showed that TiO 2 films possessed a rutile structure, with no cracks visible by atomic force microscopy (AFM), and the films morphology observed by scanning electron microscopy (SEM) was highly dependent upon the hydrothermal treatment time. Yet, the TiO 2 films with a more porous surface were more suitable for TNT-MIP film deposit. Rheology of precursor polymer film solutions, based on poly (acrylonitrile- co -acrylic acid), poly (acrylonitrile- co -methacrylic acid) or poly (acrylonitrile- co -itaconic acid), and the structure and adherence of TNT-MIP films were investigated in order to establish the correct recipe of the MIP. The removal yield of TNT from the imprinted films, the thickness, the porosity and the compatibility with the inorganic TiO 2 film were adequate for the poly (acrylonitrile- co -acrylic acid) system with an acrylonitrile: acrylic acid practical ratio of 86.1:13.9 (wt./wt.). Farmore, AFM morphology corroborated with SEM results highlighted the effect of TNT imprinting in the copolymer matrix as the surface of the imprinted layer was quite different from that of the non-imprinted layer.

Transcription factor IIH (TFIIH) is a multiprotein complex involved in both eukaryotic transcription and DNA repair, revealing a tight connection between these two processes. Composed of 10 subunits, it can be resolved into a 7-subunits core complex with the XPB translocase and the XPD helicase, and the 3-subunits kinase complex CAK, which also exists as a free complex with a distinct function. Initially identified as basal transcription factor, TFIIH also participates in transcription regulation and plays a key role in nucleotide excision repair (NER) for opening DNA at damaged sites, lesion verification and recruitment of additional repair factors. Our understanding of TFIIH function in eukaryotic cells has greatly benefited from studies of the genetic rare diseases xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD), that are not only characterized by cancer and aging predispositions but also by neurological and developmental defects. Although much remains unknown about TFIIH function, significant progresses have been done regarding the structure of the complex, the functions of its catalytic subunits and the multiple roles of the regulatory core-TFIIH subunits. This review provides a non-exhaustive survey of key discoveries on the structure and function of this pivotal factor, which can be considered as a promising target for therapeutic strategies.

Separation of naphtodianthrones (NTs) from Hypericum perforatum L. (aerial part of St. John's Wort) is still topical due to some hard-to-beat medicinal attributes of these bioactive compounds. Unfortunately, their low bioavailability (0.06%-0.4%) complicates the extraction process. Therefore, developing straightforward and lower-cost methodologies for NT separation is still a priority. In support of this purpose, for preparing NT formulations from flowers and leaves of wild St. John's Wort (hyperici herba), a cutoff preparative methodology is described herein. Combining Soxhlet extraction and reflux extraction, some concentrated and rather pure NT ethanolic-based formulations without chlorophyl and grease were obtained.

The synaptonemal complex (SC) forms during the early stages of meiotic prophase I, when it mediates the pairing of homologous chromosomes. Despite the crucial role of the SC in chromosome synapsis and genetic recombination, the molecular details of its function are still unclear. High-resolution information on the structure of SC proteins would be very valuable to elucidate the molecular basis of their function in meiosis. Here we show how cryo-electron tomography and subtomographic averaging can be usefully applied to provide insights into the structure of the helical SYCP3 protein in its filamentous state. The establishment of such method should prove of use for structural studies of other SC proteins, such as SYCP1 and the TEX12-SYCE2 complex, which can form physiologically relevant filamentous assemblies, and ultimately for the structural analysis of the SC.

In the attempt to separate pseudohypericin (PH) from Hypericum Perforatum L. (or Saint John’s Wort, SJW) phyto-extracts, molecularly imprinted polymers (MIPs) hollow beads were prepared by one-step suspension polymerisation procedures and reported herein for the first time. The main reason for preparing such hollow materials refers to enhancing the accessibility of pH to the recognition sites that further leads to selective and high rebinding yields. In this respect, various terpolymer matrixes based on methacrylic acid (MAA), acrylonitrile (AN) and ethylene glycol dimethacrylate (EDMA) were tested for the ability to form micron-sized hollow beads. Morpho-structural analyses of resulted MIP beads confirmed their hollow structure. Evaluating the influence of different parameters like solvent, pH and concentration of the feed SJW extract, upon recognition and re-binding ability of MIPs for various naphtodianthrones (NTs) from SJW phyto-extracts, reflected that the optimum rebinding concentration, specificity and selectivity for PH can be atributed to the MIP systems with 50:50 and 100:0 (wt./wt.) of MAA:AN.

This paper debates the effect of the stabilizer (vinyl benzyl trimethyl ammonium chloride—VBTAC or cetyl trimethyl ammonium chloride—CTAC) and the structure (typical SiO2 matrix or inorganic–organic interpenetrated matrix) influence in molecular imprinting of gallic acid-polyphenol class of templates. For this purpose, gallic acid-imprinted particles were obtained via typical polycondensation sol–gel reactions and via sol–gel/radical polymerization concurrent reactions of tetraethoxy silane (TEOS) and aminopropyl ethoxy silane (APTES) or vinyl triethoxy silane (VTES), respectively. The influence of the mesophase formation and stabilizer nature upon the re-binding of gallic acid was investigated using thermal, infrared and morphology measurements. Adsorption assays of organosilica particles were well correlated with their physical properties. Unlike the polycondensation of TEOS with APTES in the presence of CTAC, concurrent radical polymerization of VBTAC consumed the vinyl functionalities of VTES, decreasing the capacity of VBTAC to mediate the mesophase formation and the capacity of VTES to coordinate template molecules; hence, both the binding capacity and the “memory” effect of the interpenetrated organosilica structure were significantly affected.

In this study, composite hydrogels with interpenetrated polymer networks (IPNs), based on bacterial cellulose (BC) and poly(acrylic acid

The synaptonemal complex (SC) forms during the early stages of meiotic prophase I, when it mediates the pairing of homologous chromosomes. Despite the crucial role of the SC in chromosome synapsis and genetic recombination, the molecular details of its function are still unclear. High-resolution information on the structure of SC proteins would be very valuable to elucidate the molecular basis of their function in meiosis. Here we show how cryo-electron tomography and subtomographic averaging can be usefully applied to provide insights into the structure of the helical SYCP3 protein in its filamentous state. The establishment of such method should prove of use for structural studies of other SC proteins, such as SYCP1 and the TEX12-SYCE2 complex, which can form physiologically relevant filamentous assemblies, and ultimately for the structural analysis of the SC.

The present paper was aimed at studying the possibility of zinc (Zn) removal from the wastewater discharged from zinc electroplating processes. In order to save industrial and environmental resources, the concentrated solution could be reused after electrodialysis process. A mini-electrodialysis system with three cylindrical compartments and different membranes containing various resins (Purolite A500 and Hypersol-Macronet MN500) was employed, which can be further applied for the treatment of synthetic effluent which contained zinc ions. The electrodialysis system was operated at constant voltage using different concentrations of synthetic solutions of zinc ions, without and with electrolyte recirculation for 1.5 h. The pH and conductivity of solutions were measured before and after the electrodialysis process occurs. Also the removal ratio (Rr) and mass flow (J) of zinc ions, energy consumption (EC) and current efficiency (CE) were determined. It was found that electrodialysis treatment generated a very low conductivity solution, enabling its reuse as rinse water. According to the obtained results when using a membrane pair with higher ion exchange capacity (IEC) the removal ratio is improved (over 80%). The physico-chemical, structural and mechanical properties of prepared membranes were registered, before and after electrodialysis process takes place, by means of complementary analytical techniques, namely, ion-exchange capacity, water content and thickness measurements. Furthermore analysis were also carried out by Fourier transform infrared spectroscopy (FT-IR), environmental scanning electron microscopy (ESEM), thermal gravimetric analysis (TGA) and electrochemical impedance spectroscopy (EIS).

Novel nanocomposite hydrogel structures based on cross-linked poly(acrylic acid) (PAA) and kaolinite (Kaol), modified with different loadings of polyacrylamide (PAAm), were prepared by inverse dispersion polymerization. Ceric ammonium nitrate as an initiator in the presence of nitric acid was used to graft PAAm from the Kaol surface. The surface-modified Kaol showed enhanced interactions between the filler and the PAA matrix, through interactions between amino (–NH2) from PAAm and carboxylic groups (–COOH) from PAA. The XRD and TEM measurements confirmed the exfoliated nanocomposites with the Kaol filler. The swelling degree (SD) of the swollen hydrogel nanocomposite was increased following the addition of polyacrylamide-modified Kaol particles into the hydrogel structures. Rheological characterization showed that an increase in the storage modulus (G′) could be a consequence of a good dispersion of Kaol particles in the polyacrylic acid matrix, thereby leading to enhanced interactions and furthermore to improved mechanical properties of the final hydrogels.

In this study, the organophilization procedure of kaolin rocks with a monofunctional ethoxysilane- 3 aminopropyl dimethyl ethoxysilane (APMS) is depicted for the first time. The two-step organophilization procedure, including dimethyl sulfoxide intercalation and APMS grafting onto the inner hydroxyl surface of kaolinite (the mineral) layers was tested for three sources of kaolin rocks (KR, KC and KD) with various morphologies and kaolinite compositions. The load of APMS in the kaolinite interlayer space was higher than that of 3-aminopropyl triethoxysilane (APTS) due to the single-point grafting nature of the organophilization reaction. A higher long-distance order of kaolinite layers with low staking was obtained for the APMS, due to a more controllable organiphilization reaction. Last but not least, the solid state (29)Si-NMR tests confirmed the single-point grafting mechanism of APMS, corroborating monodentate fixation on the kaolinite hydroxyl facets, with no contribution to the bidentate or tridentate fixation as observed for APTS.

The host–guest radical polymerization of vinyl acetate in the inorganic porous matrix improved both, thermal and mechanical properties of final modified material, relative to the polymer alone, which can be further used for various applications where stability in aggressive environments is required. The bulk material was characterized by various techniques i.e. elemental analysis, X-ray photoelectron spectroscopy (XPS), gel permeation chromatography (GPC), thermal gravimetric analysis (TGA), dynamic mechanical analysis (DMA), scanning electron microscopy (SEM) and porosity measurements (BET). XPS measurements indicated a preferential order of acetate groups at the surface of the hybrid and BET analyses highlighted the formation of a new porous structure derived from the mesoporous inorganic host. GPC results of the polymer extracted from the zeolite matrix showed variations of molecular weight and low polydispersity index relative to the pure polymer, which could be a consequence of a controlled polymerization mechanism induced by the presence of metal ions found in the zeolite matrix. Thermal stability and elasticity of the restrained polymer compared to that of pure PVAc was notably enhanced. Char formation in the sixth degradation stage and demethylation were additional steps compared to PVAc alone indicating some modifications of the thermal degradation mechanism for the entrapped PVAc.

This study is aimed at developing an innovative approach for Indigo Carmine dye removal from synthetic solutions by electrodialysis, carried out using ion exchange membranes. The batch electrodialysis system was operated at various current intensities: 0.05, 0.1 and 0.15 A. The pH and conductivity of solutions were measured before and after using electrodialysis process. The colour removal efficiency (CR %) was determined by spectrographic analysis and the energy consumption (EC) was calculated. The obtained results show that the pH of treated solution increases due to the increase in solution conductivity. Moreover, the values of CR % and EC increase when increasing current intensity. The optimal value was obtained at 0.15 A (CR > 97%). The membranes were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis and scanning electron microscopy.

We performed a study in 102 people with type 2 diabetes aiming to determine "easy-to-use" predictive factors for glycemic response to glitazones. We found that low baseline HDL-cholesterol (

The main components of Orthosiphon stamineus Benth. leaves and extracts are the pharmacologically active polyphenols: the polymethoxylated flavonoids and the caffeic acid derivatives. Two tinctures, having different alcohol concentration, were studied from phytochemical and pharmacological point of view. The main polyphenols were identified and quantitatively determined by HPLC. Comparison of the retention parameters and UV-Vis spectra of standards and those of the separated compounds performed the identification of caffeic-, cichoric- and rosmarinic acids, respectively, of sinesetine and eupatorine. The quantitative determination was performed by external standard method. The diuretic, saluretic and uricosuric actions of the studied tinctures were compared by experiments on rats.

The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

The main goal of this study was to explore the suitability and performance of bicomponent polymer membranes based on acrylonitrile copolymers-polyvinyl alcohol (PVA) mixtures and small quantities of anion (Purolite A100) or cation exchange resin (Purolite C150), prepared by phase inversion. Membranes were used for copper removal from synthetic wastewater solutions. A three detachable cylindrical compartment electrodialysis cell without recirculation of the electrolytes and synthetic solutions of various concentrations, similar to a copper electrowinning electrolyte, were used. The electrodialysis unit operates under galvanostatic control. The effect of pH on electrodialysis separation of Cu2+ and on the solution conductivity has been also investigated. The laboratory electrodialysis cell performance was evaluated in terms of percent of extraction (pe) and current efficiency (ce). Experimental results showed that the ionic transfer in electrodialysis cell was especially affected by concentration. The highest values for the pe (>81 %) and the ce (>25 %) of copper ions were obtained at maximum concentration in copper ions (3 g/L), indicating a better performance of the ion extraction. The transport of copper ions was also correlated with flux data. The ion exchange membranes were characterized using FT-IR spectroscopy, ESEM, and electrochemical impedance spectroscopy.

Transcription factor IIH (TFIIH) is a eukaryotic multicomponent macromolecular assembly composed of 10 subunits including two deoxyribonucleic acid (DNA)-dependent helicase activities of opposite polarities and a kinase. Mutations in the helicases xeroderma pigmentosum group D protein (XPD) and xeroderma pigmentosum group B protein (XPB) as well as in the p8/TTD-A subunit of human TFIIH are associated with three dramatic genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). Initially recognised as a basal factor responsible for transcription initiation of protein coding genes and transition from initiation to elongation, TFIIH also plays a critical role in nucleotide excision repair via its DNA-opening helicase activity at the site of a lesion. A subcomplex, harbouring its kinase activity, phosphorylates the carboxy-terminal domain (CTD) of ribonucleic acid (RNA) polymerase II as well as a number of transcription regulators including nuclear hormone receptors. Key Concepts: TFIIH is a prime example of multienzyme multitasking macromolecular assembly. Recent data suggest that several components are found in nonTFIIH complexes and that the function of TFIIH is regulated by its subunit composition. The human transcription/DNA repair factor TFIIH is a multi-subunit complex composed two functional and structural entities: core-TFIIH consists of XPB, p62, p52, p44, p34 and p8, whereas the CDK-activating kinase (CAK) subcomplex contains cyclin-dependent kinase 7 (CDK7), cyclin H and MAT1. XPD bridges the core-TFIIH to the CDK activating kinase (CAK) complex, which also exists as a free complex with a distinct function. Although identified as a basal factor responsible for transcription initiation, TFIIH also plays a critical role in nucleotide excision repair and is implicated in the control of cell cycle progression. Key insights into TFIIH result from investigations centred on the analysis of mutations identified in XP, CS and TTD patients. Most mutations identified in patients reside within the noncatalytic regions of the helicases and do not affect the enzymatic activity of TFIIH per se but rather disturb the interactions of the catalytic subunits with their regulatory partners, impairing transcription and DNA repair. Keywords: transcription; DNA repair; helicase; xeroderma pigmentosum; trichothiodystrophy

International audience; The xeroderma pigmentosum group D (XPD) helicase is a subunit of transcription/DNA repair factor, transcription factor II H (TFIIH) that catalyzes the unwinding of a damaged DNA duplex during nucleotide excision repair. Apart from two canonical helicase domains, XPD is composed of a 4Fe-S cluster domain involved in DNA damage recognition and a module of uncharacterized function termed the “ARCH domain.” By investigating the consequences of a mutation found in a patient with trichothiodystrophy, we show that the ARCH domain is critical for the recruitment of the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. Indeed, this mutation not only affects the interaction with the MAT1 CAK subunit, thereby decreasing the in vitro basal transcription activity of TFIIH itself and impeding the efficient recruitment of the transcription machinery on the promoter of an activated gene, but also impairs the DNA unwinding activity of XPD and the nucleotide excision repair activity of TFIIH. We further demonstrate the role of CAK in downregulating the XPD helicase activity within TFIIH. Taken together, our results identify the ARCH domain of XPD as a platform for the recruitment of CAK and as a potential molecular switch that might control TFIIH composition and play a key role in the conversion of TFIIH from a factor active in transcription to a factor involved in DNA repair.

Centrins are calcium binding proteins that belong to the EF-hand (or calmodulin) superfamily, which are highly conserved among eukaryotes. Herein, we report the molecular features and binding properties of the green alga Scherffelia dubia centrin (SdCen), a member of the Chlamydomonas reinhardtii centrin (CrCen) subfamily. The Ca(2+) binding capacity of SdCen and its isolated N- and C-terminal domains (N-SdCen and C-SdCen, respectively) was investigated using flow dialysis and isothermal titration calorimetry. In contrast with human centrin 1 and 2 (from the same subfamily), but like CrCen, SdCen exhibits three physiologically significant Ca(2+) binding sites, two in the N-terminal domain and one in the C-terminal domain. Mg(2+) ions could compete with Ca(2+) in one of the N-terminal sites. When Ca(2+) binds, the N-terminal domain becomes more stable and exposes a significant hydrophobic surface that binds hydrophobic fluorescent probes. The Ca(2+) binding properties and the metal ion-induced structural changes in the C-terminal domain are comparable to those of human centrins. We used isothermal titration calorimetry to quantify the binding of SdCen, N-SdCen, and C-SdCen to three types of natural target peptides, derived from the human XPC protein (P17-XPC), the human Sfi1 protein (R17-hSfi1), and the yeast Kar1 protein (P19-Kar1). The three peptides possess the complete (P17-XPC and R17-hSfi1) or partial (P19-Kar1) centrin binding motif (W(1)L(4)L(8)). The integral SdCen exhibits two binding sites for each target peptide, with distinct affinities for each site and each peptide. The high-affinity peptide binding site corresponds to the C-terminal domain of SdCen and displays binding constants and the poor Ca(2+) sensitivities similar to those observed for human centrins. The low-affinity site constituted by the N-terminal domain is active only in the presence of Ca(2+). The thermodynamic binding parameters suggest that the C-terminal domain of SdCen may be constitutively bound to a target, while the N-terminal domain could bind a target only after a Ca(2+) signal. SdCen is also able to interact with calmodulin binding peptides (W(1)F(5)V(8)F(14) motif) with a 1:1 stoichiometry, whereas the isolated N- and C-terminal domains have a much lower affinity. These data suggest particular molecular mechanisms used by SdCen (and probably by other algal centrins) to respond to cellular Ca(2+) signals.