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Epidemiological studies inversely associate BMI with breast cancer risk in premenopausal women, but the pathophysiological linkage remains ill-defined. Despite the documented relevance of the ‘local’ environment to breast cancer progression and the well-accepted differences in transcriptome and metabolic properties of anatomically distinct fat depots, specific breast adipose contributions to the proliferative potential of non-diseased breast glandular compartment are not fully understood. To address early breast cancer causation in the context of obesity status, we compared the cellular and molecular phenotypes of breast adipose and matched breast glandular tissue from premenopausal non-obese (mean BMI = 27 kg/m2) and obese (mean BMI = 44 kg/m2) women. Breast adipose from obese women showed higher expression levels of adipogenic, pro-inflammatory, and estrogen synthetic genes than from non-obese women. Obese breast glandular tissue displayed lower proliferation and inflammatory status and higher expression of anti-proliferative/pro-senescence biomarkers TP53 and p21 than from non-obese women. Transcript levels for T-cell receptor and co-receptors CD3 and CD4 were higher in breast adipose of obese cohorts, coincident with elevated adipose interleukin 10 (IL10) and FOXP3 gene expression. In human breast epithelial cell lines MCF10A and HMEC, recombinant human IL10 reduced cell viability and CCND1 transcript levels, increased those of TP53 and p21, and promoted (MCF10A) apoptosis. Our findings suggest that breast adipose-associated IL10 may mediate paracrine interactions between non-diseased breast adipose and breast glandular compartments and highlight how breast adipose may program the local inflammatory milieu, partly by recruiting FOXP3+ T regulatory cells, to influence premenopausal breast cancer risk.

Pectinases are widely used in a variety of industrial processes. However, their application is limited by low catalytic processivity, reduced stability, high cost, and poor re-use compatibility. These drawbacks may be overcome by enzyme immobilization with ferromagnetic nanoparticles, which are easily recovered by a magnetic field. In this work, an endopolygalacturonase from Chondrostereum purpureum (EndoPGCp) expressed in Pichia pastoris was immobilized on glutaraldehyde-activated chitosan ferromagnetic nanoparticles (EndoPGCp-MNP) and used to supplement a commercial enzyme cocktail. No significant differences in biochemical and kinetic properties were observed between EndoPGCp-MNP and EndoPGCp, although the EndoPGCp-MNP showed slightly increased thermostability. Cocktail supplementation with EndoPGCp-MNP increased reducing sugar release from orange wastes by 1.8-fold and showed a synergistic effect as compared to the free enzyme. Furthermore, EndoPGCp-MNP retained 65% of the initial activity after 7 cycles of re-use. These properties suggest that EndoPGCp-MNP may find applications in the processing of pectin-rich agroindustrial residues.

The C-terminal of G protein-coupled receptors is now recognized as being important for G protein activation and signaling function. To detect the role of C-terminal tail in receptor activation, we used the α1b-AR, which has a long C-terminal of 164 amino acids. We constructed the intramolecular FRET sensors, in which the C-terminal was truncated to 10 (∆C-10), 20 (∆C-20), 30 (∆C-30), 50 (∆C-50), 70 (∆C-70), or 90 (∆C-90). The truncated mutants of ∆C-10, ∆C-20, or ∆C-30 cannot induce FRET signal changes and downstream ERK1/2 phosphorylation. However, the truncated mutants of ∆C-50, ∆C-70, or ∆C-90 induce significant FRET signal changes and downstream ERK1/2 phosphorylation, especially ∆C-90. This is particularly true in the case of the ∆C-90, ∆C-70, or ∆C-50 which retained the potential phosphorylation sites (Ser401, Ser404, Ser408, or Ser410). The ∆C-90 showed an increase in agonist-induced FRET signal changes and ERK1/2 phosphorylation in PKC- or endocytosis-dependent and EGFR-, src-, or β-arrestin2-independent.

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M 1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M 1 and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M 1 -mEGFP was expressed at levels equal to the M 1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M 1 -mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M 1 -mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M 1 -mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

Ferulic acid has antioxidant properties of interest to the food industry and can be released from natural plant fibres using feruloyl esterases. Esterases active at high temperatures are highly desirable but currently underrepresented. Here we report the biochemical characterization of the feruloyl esterase from Thermobacillus xylanilyticus. Specific activity of recombinant Tx-Est1 with ethyl ferulate was 29.2 ± 2.9 U mg

Endoglucanases (EC 3.2.1.4) are important enzymes involved in the hydrolysis of cellulose, acting randomly in the β-1,4-glycosidic bonds present in the amorphous regions of the polysaccharide chain. These biocatalysts have been classified into 14 glycosyl hydrolase (GH) families. The GH7 family is of particular interest since it may act on a broad range of substrates, including cellulose, β-glucan, and xylan, an attractive feature for biotechnological applications, especially in the renewable energy field. In the current work, a gene from the thermophilic fungus Thermothielavioides terrestris, encoding an endoglucanase GH7 (TtCel7B), was cloned in the secretion vector pEXPYR and transformed into the high-protein-producing strain Aspergillus nidulans A773. Purified TtCel7B has a molecular weight of approximately 66 kDa, evidenced by SDS-PAGE. Circular dichroism confirmed the high β-strand content consistent with the canonical GH7 family β-jellyroll fold, also observed in the 3D homology model of TtCel7B. Biochemical characterization assays showed that TtCel7B was active over a wide range of pH values (3.5–7.0) and temperatures (45–70 °C), with the highest activity at pH 4.0 and 65 °C. TtCel7B also was stable over a wide range of pH values (3.5–9.0), maintaining more than 80% of its activity after 24 h. The KM and Vmax values in low-viscosity carboxymethylcellulose were 9.3 mg mL−1 and 2.5 × 104 U mg−1, respectively. The results obtained in this work provide a basis for the development of applications of recombinant TtCel7B in the renewable energy field.

A new strain of Trichoderma reesei (teleomorph Hypocrea jecorina) with high cellulase production was obtained by exposing the spores from T. reesei QM9414 to an ultraviolet light followed by selecting fast-growing colonies on plates containing CMC (1% w/v) as the carbon source. The mutant T. reesei RP698 reduced cultivation period to 5 days and increased tolerance to the end-products of enzymatic cellulose digestion. Under submerged fermentation conditions, FPase, CMCase, and Avicelase production increased up to 2-fold as compared to the original QM9414 strain. The highest levels of cellulase activity were obtained at 27 °C after 72 h with Avicel®, cellobiose, and sugarcane bagasse as carbon sources. The temperature and pH activity optima of the FPase, CMCase, and Avicelase were approximately 60 °C and 5.0, respectively. The cellulase activity was unaffected by the addition of 140 mM glucose in the enzyme assay. When T. reesei RP698 crude extract was supplemented by the addition of β-glucosidase from Scytalidium thermophilum, a 2.3-fold increase in glucose release was observed, confirming the low inhibition by the end-product of cellulose hydrolysis. These features indicate the utility of this mutant strain in the production of enzymatic cocktails for biomass degradation.

Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein–protein interactions.

Graphical abstract, Highlights • A left-handed triangular prism beta-solenoid scaffold (BeSS) was designed, built and validated. • Vertex inserts in BeSS-CBM3-GH11 xylanase chimeras varies interdomain distance and orientation. • A library of BeSS-CBM3-GH11 chimeras tested against various lignocellulosic biomasses. • Maximum reducing sugar release when CBM3 and GH11 domains aligned on the same vertex. • CBM3 binding to cellulose fibres facilitates GH11 hydrolysis of intercalated xylan., Effective use of plant biomass as an abundant and renewable feedstock for biofuel production and biorefinery requires efficient enzymatic mobilization of cell wall polymers. Knowledge of plant cell wall composition and architecture has been exploited to develop novel multifunctional enzymes with improved activity against lignocellulose, where a left-handed β–3-prism synthetic scaffold (BeSS) was designed for insertion of multiple protein domains at the prism vertices. This allowed construction of a series of chimeras fusing variable numbers of a GH11 β-endo-1,4-xylanase and the CipA-CBM3 with defined distances and constrained relative orientations between catalytic domains. The cellulose binding and endoxylanase activities of all chimeras were maintained. Activity against lignocellulose substrates revealed a rapid 1.6- to 3-fold increase in total reducing saccharide release and increased levels of all major oligosaccharides as measured by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). A construct with CBM3 and GH11 domains inserted in the same prism vertex showed highest activity, demonstrating interdomain geometry rather than number of catalytic sites is important for optimized chimera design. These results confirm that the BeSS concept is robust and can be successfully applied to the construction of multifunctional chimeras, which expands the possibilities for knowledge-based protein design.

CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested that the receptor may exist as a dimer or an oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means, we confirm that CXCR4 can exist as a mixture of monomers, dimers, and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t-a small drug-like isothiourea derivative-rapidly destabilizes the oligomeric structure, whereas AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.

The regulation of the translation of messenger RNA (mRNA) in eukaryotic cells is critical for gene expression, and occurs principally at the initiation phase which is mainly regulated by eukaryotic initiation factors (eIFs). eIFs are fundamental for the translation of mRNA and as such act as the primary targets of several signaling pathways to regulate gene expression. Mis-regulated mRNA expression is a common feature of tumorigenesis and the abnormal activity of eIF complexes triggered by upstream signaling pathways is detected in many tumors, leading to the selective translation of mRNA encoding proteins involved in tumorigenesis, metastasis, or resistance to anti-cancer drugs, and making eIFs a promising therapeutic target for various types of cancers. Here, we briefly outline our current understanding of the biology of eIFs, mainly focusing on the effects of several signaling pathways upon their functions and discuss their contributions to the initiation and progression of tumor growth. An overview of the progress in developing agents targeting the components of translation machinery for cancer treatment is also provided. Video abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12964-020-00607-9.

Background: Dictamnine (Dic), a naturally occurring furoquinoline alkaloid isolated from the root bark of Dictamnus dasycarpus Turcz., is reported to display a wide range of potential pharmacological properties including anticancer activity against multiple cancer types. However, little is known about the direct target proteins and anticancer mechanisms of Dic.Methods: Anticancer effects of Dic and chemotherapy resistance of lung cancer were determined by CCK8, EdU and apoptosis assay. Boyden chamber migration and invasion, wound healing assay, plate colony formation and sphere formation assay were performed to explore the effects of Dic on metastasis and stemness of lung cancer cells. Protein docking analysis, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) were used for prediction and confirmation of the interaction between Dic and c-Met. qRT-PCR, Western blotting and immunohistochemistry (IHC) were used in mechanism investigation. Tumor xenograft model was used to evaluate the anti-tumor effects of Dic in vivo.Results: Dic was found to suppress the proliferation of lung cancer cells and attenuate the activation of the PI3K/AKT/mTOR and mitogen-activated protein kinase (MAPK) signaling pathways by directly inhibiting the phosphorylation and activation of the receptor tyrosine kinase c-Met. Moreover, Dic treatment significantly inhibited the colony formation, migration, invasion, stemness, adhesive ability, epithelial-mesenchymal transition and in vivo xenograft tumor growth of A549 lung cancer cells. Notably, the combination of Dic and gefitinib synergistically inhibited the cell proliferation, induced apoptosis, and suppressed the PI3K/Akt/mTOR and MAPK signaling pathways in PC9 gefitinib resistant cells.Conclusions: In conclusion, Dic was identified as a novel c-Met inhibitor and our results suggest the potential use of Dic as a new therapeutic agent in the treatment of lung cancer or other cancers with overactive c-Met pathway.

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein–coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography–mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P–NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P–binding receptor via liquid chromatography–tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.

β-glucosidases catalyze the hydrolysis β-1,4, β-1,3 and β-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl β-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) β-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source. Kinetic characterization revealed that the MpBgl3 was highly tolerant to glucose, which is in contrast to many Bgls that are completely inhibited by glucose. A 3D model of MpBgl3 was generated by molecular modeling and used for the evaluation of structural differences with a Bgl3 that is inhibited by glucose. Taken together, our results provide new clues to understand the glucose tolerance in GH3 β-glucosidases.

Microbial β-galactosidases (EC 3.1.2.23) have applications in the production of galacto-oligosaccharides, which are established prebiotic food ingredients. The β-galactosidase from Bacillus subtilis (YesZ) was expressed as a heterologous protein in Escherichia coli, and presented an optimum activity at pH 6.5 and 40 °C. The catalytic constants Km and Vmax of the enzyme were 8.26 mM and 1.42 μmol·min−1·mg−1 against pNP-β- d -galactopyranoside, respectively. Structural characterization revealed that YesZ is a homotrimer in solution, and homology modeling suggested that the YesZ conserves a Cys cluster zinc binding site. Flame photometry experiments confirmed the presence of bound zinc in the recombinant enzyme, and YesZ activity was inhibited by 1 mM zinc, copper and silver ions. Transgalactosylation activity of YesZ was observed with the synthetic substrate p-NP-βGal in the presence of a d -xylose acceptor, producing a β- d -galactopyranosyl-(1 → 4)- d -xylopyranose disaccharide. Analysis of this disaccharide by MALDI-ToF-MS/MS suggested a β-1,4 glycosidic linkage between a non-reducing galactose residue and the xylose. The β-galactosidase YesZ from B. subtilis is a candidate for enzymatic synthesis showing favorable thermostability (with residual activity of 50% after incubation at 30 °C for 25 h) and transgalactosylation activity.

The cannabinoid receptor 1 (CB1) is mainly expressed in the nervous system and regulates learning, memory processes, pain and energy metabolism. However, there is no way to directly measure its activation. In this study, we constructed a CB1 intramolecular fluorescence resonance energy transfer (FRET) sensor, which could measure CB1 activation by monitoring structural changes between the third intracellular loop and the C-terminal tail. CB1 agonists induced a time- and concentration-dependent increase in the FRET signal, corresponding to a reduction in the distance between the third intracellular loop and the C-terminal tail. This, in turn, mobilized intracellular Ca2+, inhibited cAMP accumulation, and increased phosphorylation of the ERK1/2 MAP kinases. The activation kinetics detected using this method were consistent with those from previous reports. Moreover, the increased FRET signal was markedly inhibited by the CB1 antagonist rimonabant, which also reduced phosphorylation of the ERK1/2 MAP kinases. We mutated a single cysteine residue in the sensor (at position 257 or 264) to alanine. Both mutation reduced the agonist-induced increase in FRET signal and structural changes in the CB1 receptor, which attenuated phosphorylation of the ERK1/2 MAP kinases. In summary, our sensor directly assesses the kinetics of CB1 activation in real-time and can be used to monitor CB1 structure and function.

Activation of the histamine-3 receptor (H3R) is involved in memory processes and cognitive action, while blocking H3R activation can slow the progression of neurological disorders, such as Alzheimer's disease, schizophrenia and narcolepsy. To date, however, no direct way to examine the activation of H3R has been utilized. Here, we describe a novel biosensor that can visualize the activation of H3R through an intramolecular fluorescence resonance energy transfer (FRET) signal. To achieve this, we constructed an intramolecular H3R FRET sensor with cyan fluorescent protein (CFP) attached at the C terminus and yellow fluorescent protein (YFP) inserted into the third intracellular loop. The sensor was found to internalize normally on agonist treatment. We measured FRET signals between the donor CFP and the acceptor YFP in living cells in real time, the results of which indicated that H3R agonist treatment (imetit or histamine) increases the FRET signal in a time- and concentration-dependent manner with Kon and Koff values consistent with published data and which maybe correlated with decreasing cAMP levels and the promotion of ERK1/2 phosphorylation. The FRET signal was inhibited by H3R antagonists, and the introduction of mutations at F419A, F423A, L426A and L427A, once again, the promotion of ERK1/2 phosphorylation, was diminished. Thus, we have built a H3R biosensor which can visualize the activation of receptor through real-time structure changes and which can obtain pharmacological kinetic data at the same time. The FRET signals may allow the sensor to become a useful tool for screening compounds and optimizing useful ligands.

The β- d -glucans are abundant cell wall polysaccharides in many cereals and contain both (1,3)- and (1,4)-bonds. The β-1,3–1,4-glucanases (EC 3.2.1.73) hydrolyze β-(1,4)- d -glucosidic linkages in glucans, and have applications in both animal and human food industries. A chimera between the family 11 carbohydrate-binding module from Ruminoclostridium (Clostridium) thermocellum celH (RtCBM11), with the β-1,3–1,4-glucanase from Bacillus subtilis (BglS) was constructed by end-to-end fusion (RtCBM11-BglS) to evaluate the effects on the catalytic function and its application in barley β-glucan degradation for the brewing industry. The parental and chimeric BglS presented the same optimum pH (6.0) and temperature (50 °C) for maximum activity. The RtCBM11-BglS showed increased thermal stability and 30% higher hydrolytic efficiency against purified barley β-glucan, and the rate of hydrolysis of β-1,3–1,4-glucan in crude barley extracts was significantly increased. The enhanced catalytic performance of the RtCBM11-BglS may be useful for the treatment of crude barley extracts in the brewing industry.

Enzyme reaction products and by-products from pretreatment steps can inhibit endoglucanases and are major factors limiting the efficiency of enzymatic lignocellulosic biomass hydrolysis. The gene encoding the endoglucanase from Scytalidium thermophilum (egst) was cloned and expressed as a soluble protein in Pichia pastoris GS115. The recombinant enzyme (Egst) was monomeric (66 kDa) and showed an estimated carbohydrate content of 53.3% (w/w). The optimum temperature and pH of catalysis were 60–70 °C and pH of 5.5, respectively. The enzyme was highly stable at pH 3.0–8.0 with a half-life in water of 100 min at 65 °C. The Egst presented good halotolerance, retaining 84.1 and 71.4% of the control activity in the presence of 0.5 and 2.0 mol L−1 NaCl, respectively. Hydrolysis of medium viscosity carboxymethylcellulose (CMC) by Egst was stimulated 1.77-, 1.84-, 1.64-, and 1.8-fold by dithiothreitol, β-mercaptoethanol, cysteine, and manganese at 10, 10, 10, and 5 mmol L−1 concentration, respectively. The enzyme hydrolyzed CMC with maximal velocity and an apparent affinity constant of 432.10 ± 16.76 and 10.5 ± 2.53 mg mL−1, respectively. Furthermore, the Egst was tolerant to reaction products and able to act on pretreated fractions sugarcane bagasse demonstrating excellent properties for application in the hydrolysis of lignocellulosic biomass.

Made available in DSpace on 2020-12-10T17:29:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The application of enzymes for sustainable and low-environmental impact industrial processes requires high-level enzyme production at low-cost. A promising strategy is the use of a high efficiency heterologous protein expression system using E. coli and the pT7BsXA vector encoding the GH11 xylanase from Bacillus subtilis with promoter, replication origin and signal peptide sequences from B. subtilis (Ruller et al. 2006). This expression system produces high amounts of enzyme that are secreted to the culture broth. The present study aimed to maximize the xylanase production by this system through evaluation of culture medium composition. Different culture media previously described in the literature together with compositions derived from agro-industrial residues were evaluated. A culture medium derived from agro-industrial residues using sugarcane molasses as carbon source showed a 9-fold increase in enzyme production (195,000 U/L) in relation to LB medium and the lowest production cost, which was 8.5-fold lower than LB medium using sugarcane molasses as carbon source and brewer's yeast as vitamin source in shaker experiments. In a bioreactor experiment the best production medium promoted an 8.5-fold higher production at a 10.8-fold lower cost as compared to shaker LB cultivation. Verdartis Desenvolvimento Biotecnol Ltda, Av Dra Naldir Ribeirao Preto,1805,Jardim Dr Paulo, BR-14056680 Ribeirao Preto, SP, Brazil Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, Ave Bandeirantes 3-900, BR-14040900 Ribeirao Preto, SP, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut, Dept Bioprocessos & Biotecnol, Rodovia Araraquara Jau,Km 01,Campos Ville, BR-14800903 Araraquara, SP, Brazil Fundacao Oswaldo Cruz, FIOCRUZ, Rua Sao Jose S-N, BR-61760000 Eusebio, CE, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut, Dept Bioprocessos & Biotecnol, Rodovia Araraquara Jau,Km 01,Campos Ville, BR-14800903 Araraquara, SP, Brazil FAPESP: 2008/53426-7 FAPESP: 2010/50328-4

The use of non-potable water (such as seawater) is an attractive alternative for water intensive processes such as biomass pretreatment and saccharification steps in the production of biochemicals and biofuels. Identification and application of halotolerant enzymes compatible with high-salt conditions may reduce the energy needed for non-potable water treatment and decrease waste treatment costs. Here we present the biochemical properties of a halotolerant endo-1,4-β-xylanase produced by Aspergillus clavatus in submerged fermentation, using paper sludge (XPS) and sugarcane bagasse (XSCB), and its potential application in the hydrolysis of agroindustrial residues. The peptide mass fingerprint and amino acid sequencing of the XPS and XSCB enzymes showed primary structure similarities with an endo-1,4-β-xylanase from Aspergillus clavatus (XYNA_ASPCL). Both enzyme preparations presented good thermal stability at 50 °C and were stable over a wide range of pH and Vmax up to 2450 U/mg for XPS. XPS and XSCB were almost fully stable even after 24 h of incubation in the presence of up to 3 M NaCl, and their activity were not affected by 500 mM NaCl. Both enzyme preparations were capable of hydrolyzing paper sludge and sugarcane bagasse to release reducing sugars. These characteristics make this xylanase attractive to be used in the hydrolysis of biomass, particularly with brackish water or seawater.

Current methods for determining membrane protein association in cells are either very time consuming, require complicated procedures, or lack the sensitivity needed to assess the effect of concentration or ligand binding on the observed oligomerization. To overcome these limitations, we provide a detailed protocol for quantifying the relative abundance and stability of oligomers of differing sizes using two-dimensional fluorescence intensity fluctuation (2D-FIF) spectrometry. The approach can be implemented using a standard laser scanning fluorescence microscope along with custom written software for image analysis. This method may be applied to evaluate the extent of oligomerization as a function of receptor concentration and is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest.

Endopolygalacturonase (EndoPG) from Stereum purpureum was expressed as a soluble protein in Pichia pastoris GS115, where after 3 days methanol induction the enzyme activity in the culture supernatant was 40 U mL−1. After purification by IMAC, SDS–PAGE analysis showed that the molecular weight of EndoPG was approximately 60.0 kDa. The carbohydrate content of the recombinant enzyme was estimated to be 67.0% (w/w). The optimum temperature and pH of catalysis were 60–70 °C and pH of 4.5, respectively. The enzyme was highly stable over the pH range 6.0–8.0 and retained approximately 60% of its initial activity after incubation at 70 °C for 30 min. The enzyme showed a specific activity of 5040.0 ± 217 U mg−1 and hydrolyzed citrus pectin with Vmax and a KM of 4947.10 ± 393.63 U mg−1 and 2.45 ± 0.23 mg mL−1, respectively, and showed a catalytic efficiency of 2052.90 ± 193.54 mL mg−1 s−1. EndoPG alone reduced the viscosity of papaya juice by 20% after 30 min, and increased its transmittance about 50% with a concomitant reduction of the color by about 55% after 5 h of enzymatic treatment. For apple juice, the relative reduction of viscosity was 30% after 5 h, and the reduction of the color was 30% with a 12% increase in transmittance. Supplementation of a commercial enzymatic cocktail for lignocellulose saccharification with EndoPG increased total reducing sugar release by 8.6 ± 2.1% against sugar cane bagasse, indicating improved access of the cellulolytic enzymes to the biomass polysaccharides.

Graphical abstract Cartoon depictions of (a) Dynamic interchange between receptor monomers, (homo, orange GPCR1, and hetero, orange GPCR1 and green GPCR2) dimers and oligomers which may be ligand regulated (orange sphere). (b) Signalling cross-talk between protomers of a dimer. Left: ligand (orange sphere) binds to one protomer and the conformational change transmitted to the second protomer results in signalling through activation of a Gα subunit (blue). Right: ligand (green sphere) binding to the second protomer may modulate signalling (large or small yellow flash)., G protein-coupled receptors (GPCRs) are an extensive class of trans-plasma membrane proteins that function to regulate a wide range of physiological functions. Despite a general perception that GPCRs exist as monomers an extensive literature has examined whether GPCRs can also form dimers and even higher-order oligomers, and if such organization influences various aspects of GPCR function, including cellular trafficking, ligand binding, G protein coupling and signalling. Here we focus on recent studies that employ approaches ranging from computational methods to single molecule tracking and both quantal brightness and fluorescence fluctuation measurements to assess the organization, stability and potential functional significance of dimers and oligomers within the class A, rhodopsin-like GPCR family.

G protein-coupled receptors (GPCRs) regulate an array of fundamental biological processes, such as growth, metabolism and homeostasis. Specifically, GPCRs are involved in cancer initiation and progression. However, compared with the involvement of the epidermal growth factor receptor in cancer, that of GPCRs have been largely ignored. Recent findings have implicated many GPCRs in tumorigenesis, tumor progression, invasion and metastasis. Moreover, GPCRs contribute to the establishment and maintenance of a microenvironment which is permissive for tumor formation and growth, including effects upon surrounding blood vessels, signaling molecules and the extracellular matrix. Thus, GPCRs are considered to be among the most useful drug targets against many solid cancers. Development of selective ligands targeting GPCRs may provide novel and effective treatment strategies against cancer and some anticancer compounds are now in clinical trials. Here, we focus on tumor related GPCRs, such as G protein-coupled receptor 30, the lysophosphatidic acid receptor, angiotensin receptors 1 and 2, the sphingosine 1-phosphate receptors and gastrin releasing peptide receptor. We also summarize their tissue distributions, activation and roles in tumorigenesis and discuss the potential use of GPCR agonists and antagonists in cancer therapy.

Current technologies for probing membrane protein association and stability in cells are either very laborious or lack the bandwidth needed for fully quantitative analysis. Here we introduce a platform, termedone-ortwo-dimensional fluorescence intensity fluctuation spectrometry, for determining the identity, abundance, and stability of oligomers of differing sizes. The sensitivity of this approach is demonstrated by using monomers and oligomers of known sizes in both solutions and cell membranes. The analysis was extended to uncover the oligomeric states and their stability for both the epidermal growth factor receptor, a receptor tyrosine kinase, and the G protein-coupled secretin receptor. In both cases, agonist ligand binding shifted the equilibrium from monomers or dimers to rather large oligomers. Our method can be used in conjunction with a variety of light-based microscopy techniques, is several orders of magnitude faster than current approaches, and is scalable for high-throughput analyses.

Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein-protein interactions.

Nanoparticles can act as support materials for enzymatic immobilization, introducing a balance of characteristics that modulate the efficiency of biocatalysts, such as specific surface area, resistance to mass transfer and effective enzymatic loading. Magnetic nanoparticles can be easily separated using an external magnetic field, and in this work two recombinant enzymes, the β-glucosidase from Humicola insolens (Bglhi) and the endoglucanase from Scytalidium thermophilum (Egst) were immobilized on synthetized Fe3O4 nanoparticles derivatized with chitosan/glutaraldehyde/N-(5-amino-1-carboxy-pentyl) iminodiacetic acid and functionalized with NiCl2. The immobilization yields were about 20% for Bglhi and Egst with efficiencies of 132% and 115%, respectively. The two enzymes were also co-immobilized with yield was about 49%. The optimal temperatures of the immobilized enzymes were 70 °C and 55 °C for Egst and Bglhi, respectively. Egst hydrolyzed CMC in the presence of 4 mM MnCl2 with Vmax = 625.0 ± 6.7 U mg−1 and KM = 6.4 ± 0.5 mg mL−1 resulting in a catalytic efficiency (kcat/KM) of 107.4 ± 5.4 mg−1 s−1 mL. Bglhi hydrolyzed pNP-Glc with Vmax = 52.7 ± 2.7 U mg−1 and KM = 0.23 ± 0.01 mM resulting in a catalytic efficiency (kcat/KM) of 214.3 ± 10.2 s−1 mM−1. The individually immobilized enzymes when combined showed a synergistic effect on the substrates tested and a very similar action when compared to the co-immobilized enzymes, suggesting excellent potential for application in biotechnological processes.

Carbohydrate binding modules (CBMs) are non-catalytic domains within larger multidomain polypeptides. The CelH from Ruminoclostridium (Clostridium) thermocellum contains a family 11 CBM (RtCBM11) with high binding affinity for the linear polysaccharide β-glucan, and low affinity for the branched xyloglucan. Screening a random RtCBM11 mutant phage library created by error prone PCR for xyloglucan binding identified RtCBM11 mutants with enhanced xyloglucan affinity. Subsequent recombination of the selected variants by site-directed mutagenesis generated the H102L/Y152F and Y46N/G52D/H102L/Y152F mutants. Fusion of the quadruple RtCBM11 mutant with the xyloglucanase from Aspergillus niveus increased the catalytic efficiency of the enzyme by 38%. Isothermal titration calorimetry demonstrated increased xyloglucan affinity for both mutants and reduced affinity for β-glucan in the H102L/Y152F mutant. Molecular dynamics simulations indicated that the increased xyloglucan specificity results both from formation of a xylosyl binding pocket in the carbohydrate binding cleft, and via modulation of a hydrogen bond network between the oligosaccharide ligand and the protein. These results explain the improved xyloglucan binding in the RtCBM11 H102L/Y152F mutant and advance the understanding of the structural determinants of CBMs binding that discriminate between branched and unbranched polysaccharides.

Galactanases (endo-β-1,4-galactanases—EC 3.2.1.89) catalyze the hydrolysis of β-1,4 galactosidic bonds in arabinogalactan and galactan side chains found in type I rhamnogalacturan. The aim of this work was to understand the catalytic function, biophysical properties, and use of a recombinant GH53 endo-beta-1,4-galactanase for commercial cocktail supplementation. The nucleotide sequence of the endo-β-1,4-galactanase from Bacillus licheniformis CBMAI 1609 (Bl1609Gal) was cloned and expressed in Escherichia coli, and the biochemical and biophysical properties of the enzyme were characterized. The optimum pH range and temperature of Bl1609Gal activity were 6.5–8 and 40 °C, respectively. Furthermore, Bl1609Gal showed remarkable pH stability, retaining more than 75 % activity even after 24 h of incubation at pH 4–10. The enzyme was thermostable, retaining nearly 100 % activity after 1-h incubation at pH 7.0 at 25–45 °C. The enzymatic efficiency (K cat /K m ) against potato galactan under optimum conditions was 241.2 s−1 mg−1 mL. Capillary zone electrophoresis demonstrated that the pattern of galactan hydrolysis by Bl1609Gal was consistent with that of endogalactanases. Supplementation of the commercial cocktail ACCELLERASE®1500 with recombinant Bl1609Gal increased hydrolysis of pretreated sugarcane bagasse by 25 %.

The Ras-Raf-MEK-MAPK (mitogen-activated protein kinase)-signaling pathway plays a key role in the regulation of many cellular functions, including cell proliferation, differentiation and transformation, by transmitting signals from membrane receptors to various cytoplasmic and nuclear targets. One of the key components of this pathway is the serine/threonine protein kinase, Raf. The Raf family kinases (A-Raf, B-Raf and C-Raf) have been intensively studied since being identified in the early 1980s as retroviral oncogenes, especially with respect to the discovery of activating mutations of B-Raf in a large number of tumors which led to intensified efforts to develop drugs targeting Raf kinases. This also resulted in a rapid increase in our knowledge of the biological functions of the B-Raf and C-Raf isoforms, which may in turn be contrasted with the little that is known about A-Raf. The biological functions of A-Raf remain mysterious, although it appears to share some of the basic properties of the other two isoforms. Recently, emerging evidence has begun to reveal the functions of A-Raf, of which some are kinase-independent. These include the inhibition of apoptosis by binding to MST2, acting as safeguard against oncogenic transformation by suppressing extracellular signal-regulated kinases (ERK) activation and playing a role in resistance to Raf inhibitors. In this review, we discuss the regulation of A-Raf protein expression, and the roles of A-Raf in apoptosis and cancer, with a special focus on its role in resistance to Raf inhibitors. We also describe the scaffold functions of A-Raf and summarize the unexpected complexity of Raf signaling.

GPCRs (G-protein-coupled receptors) are members of a family of proteins which are generally regarded as the largest group of therapeutic drug targets. Ligands of GPCRs do not usually activate all cellular signalling pathways linked to a particular seven-transmembrane receptor in a uniform manner. The fundamental idea behind this concept is that each ligand has its own ability, while interacting with the receptor, to activate different signalling pathways (or a particular set of signalling pathways) and it is this concept which is known as biased signalling. The importance of biased signalling is that it may selectively activate biological responses to favour therapeutically beneficial signalling pathways and to avoid adverse effects. There are two levels of biased signalling. First, bias can arise from the ability of GPCRs to couple to a subset of the available G-protein subtypes: Gαs, Gαq/11, Gαi/o or Gα12/13. These subtypes produce the diverse effects of GPCRs by targeting different effectors. Secondly, biased GPCRs may differentially activate G-proteins or β-arrestins. β-Arrestins are ubiquitously expressed and function to terminate or inhibit classic G-protein signalling and initiate distinct β-arrestin-mediated signalling processes. The interplay of G-protein and β-arrestin signalling largely determines the cellular consequences of the administration of GPCR-targeted drugs. In the present review, we highlight the particular functionalities of biased signalling and discuss its biological effects subsequent to GPCR activation. We consider that biased signalling is potentially allowing a choice between signalling through ‘beneficial’ pathways and the avoidance of ‘harmful’ ones.

G protein-coupled receptors, including the M3 muscarinic acetylcholine receptor, can form homo-oligomers. However, the basis of these interactions and the overall organizational structure of such oligomers are poorly understood. Combinations of site-directed mutagenesis and homogenous time-resolved fluorescence resonance energy transfer studies that assessed interactions between receptor protomers at the surface of transfected cells indicated important contributions of regions of transmembrane domains I, IV, V, VI, and VII as well as intracellular helix VIII to the overall organization. Molecular modeling studies based on both these results and an X-ray structure of the inactive state of the M3 receptor bound by the antagonist/inverse agonist tiotropium were then employed. The results could be accommodated fully by models in which a proportion of the cell surface M3 receptor population is a tetramer with rhombic, but not linear, orientation. This is consistent with previous studies based on spectrally resolved, multiphoton fluorescence resonance energy transfer. Modeling studies furthermore suggest an important role for molecules of cholesterol at the dimer + dimer interface of the tetramer, which is consistent with the presence of cholesterol at key locations in many G protein-coupled receptor crystal structures. Mutants that displayed disrupted quaternary organization were often poorly expressed and showed immature N-glycosylation. Sustained treatment of cells expressing such mutants with the muscarinic receptor inverse agonist atropine increased cellular levels and restored both cell surface delivery and quaternary organization to many of the mutants. These observations suggest that organization as a tetramer may occur before plasma membrane delivery and may be a key step in cellular quality control assessment.

Background: The quaternary organization of G protein-coupled receptors remains a subject of considerable debate. Results: The serotonin 5-HT2C receptor is a mixture of monomers, dimers, and higher-order oligomers. These become predominantly monomers upon antagonist treatment. Conclusion: SpIDA indicates that the 5-HT2C receptor exists as mixtures of forms that are regulated in an antagonist-dependent manner. Significance: SpIDA delivers new insights into receptor quaternary structure., The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor.

Background and Aims The cultivation of dedicated biomass crops, including miscanthus, on marginal land provides a promising approach to the reduction of dependency on fossil fuels. However, little is known about the impact of environmental stresses often experienced on lower-grade agricultural land on cell-wall quality traits in miscanthus biomass crops. In this study, three different miscanthus genotypes were exposed to drought stress and nutrient stress, both separately and in combination, with the aim of evaluating their impact on plant growth and cell-wall properties. Methods Automated imaging facilities at the National Plant Phenomics Centre (NPPC-Aberystwyth) were used for dynamic phenotyping to identify plant responses to separate and combinatorial stresses. Harvested leaf and stem samples of the three miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus and Miscanthus × giganteus) were separately subjected to saccharification assays, to measure sugar release, and cell-wall composition analyses. Key Results Phenotyping showed that the M. sacchariflorus genotype Sac-5 and particularly the M. sinensis genotype Sin-11 coped better than the M. × giganteus genotype Gig-311 with drought stress when grown in nutrient-poor compost. Sugar release by enzymatic hydrolysis, used as a biomass quality measure, was significantly affected by the different environmental conditions in a stress-, genotype- and organ-dependent manner. A combination of abundant water and low nutrients resulted in the highest sugar release from leaves, while for stems this was generally associated with the combination of drought and nutrient-rich conditions. Cell-wall composition analyses suggest that changes in fine structure of cell-wall polysaccharides, including heteroxylans and pectins, possibly in association with lignin, contribute to the observed differences in cell-wall biomass sugar release. Conclusions The results highlight the importance of the assessment of miscanthus biomass quality measures in addition to biomass yield determinations and the requirement for selecting suitable miscanthus genotypes for different environmental conditions.

The β-glucosidases (β-D-glucoside glucohydrolase, EC 3.2.1.21) hydrolyze glycosidic bonds of alkyl-, amino-, or aryl-β-D-glucosides, cyanogenic glucosides, disaccharides and short oligosaccharides and can also catalyze the synthesis of glycosyl-bonds between different molecules via transglycosylation. Due to their ubiquitous phylogenetic distribution, substrate diversity and ability to both hydrolyze and synthesize glycosidic bonds, the catalysis and regulation of β-glucosidases have been extensively studied. Many β-glucosidases are inhibited by the reaction product glucose, and reduced catalytic activity may limit the biotechnological and industrial applications of these enzymes and this has stimulated the search for β-glucosidases that maintain their activity at high glucose concentrations. Studies of many glucose tolerant enzymes have been reported and due to the ongoing interest in these enzymes, here it has been reviewed this accumulated body of knowledge which provides valuable insights as to the kinetics, structure, regulation and evolution of glucose tolerant and glucose stimulated β-glucosidases.

The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 is an RNA-guided site-specific DNA-binding complex that can be reprogrammed to specifically interact with a desired DNA sequence target. CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes. Although Cas9 has been successfully used as a precise tool in all these applications, some limitations have also been reported, for instance (i) a strict dependence on a protospacer-adjacent motif (PAM) sequence, (ii) aberrant off-target activity, (iii) the large size of Cas9 is problematic for CRISPR delivery, and (iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise spatiotemporal control of gene expression or genome editing. These obstacles hinder the use of CRISPR for disease treatment and in wider biotechnological applications. Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation. Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.

A paramagnetic nanocomposite coated with chitosan and N-(5-Amino-1-carboxy-pentyl) iminodiacetic acid (NTA) that is suitable for protein immobilization applications has been prepared and characterized. The nanoparticle core was synthesized by controlled aggregation of Fe3O4 under alkaline conditions, and Transmission Electron Microscopy revealed a size distribution of 10–50 nm. The nanoparticle core was coated with chitosan and derivatized with glutaraldehyde and NTA, as confirmed by Fourier Transform Infrared Spectroscopy. The final nanoparticles were used as a metal affinity matrix to separate a recombinant polyhistidine-tagged β-galactosidase from Bacillus subtilis directly from E. coli cell lysates with high purity (>95%). After loading with Ni2+, nanoparticles demonstrated a binding capacity of 250 μg of a polyhistidine-tagged β-galactosidase per milligram of support. The immobilized enzyme retained 80% activity after 9 cycles of washing, and the immobilized recombinant protein could be eluted with high purity with imidazole. The applications for these nanomagnetic composites extend beyond protein purification, and can also be used for immobilizing enzymes, where the β-galactosidase immobilized on the nanomagnetic support was used in multiple cycles of catalytic reactions with no significant loss of catalytic activity.