Overexpression of F0F1-ATP synthase α suppresses mutant huntingtin aggregation and toxicity in vitro

Huntington’s disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As a multi-subunit protein localized in the mitochondria of eukaryotic cells, the F₀F₁-ATP synthase α belongs to the family of stress proteins HSP60. Currently, mounting evidences indicate F₀F₁-ATP synthase α may play a role in neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Recently, ATP synthase α was reported to have protective and therapeutic roles in primary cardiacmyocytes of iron-overloaded rats by lowering ROS production. However, little is understood about the role of ATP synthase α in cell death and neurodegeneration. Here, we demonstrate that overexpression of ATP synthase α suppresses huntingtin (htt) polyQ aggregation and toxicity in transfected SH-SY5Y cell lines. Overexpression of ATP synthase α is able to protect cell death caused by polyglutamine-expanded htt. Transient overexpression of ATP synthase α suppresses the aggregate formation by estimation of polyQ aggregation, Western blot analysis, and filter trap assay (FTA) in transfected SH-SY5Y cells. These results indicated that ATP synthase α has a strong inhibitory effect on polyglutamine aggregate formation and toxicity in vitro, and suggest a novel neuroprotective role of ATP synthase α.     

Kinetic mechanisms for overexpression insensitivity and oncogene cooperation

Minor (5-10 fold) activation of mitogenic signalling cascades typically induces cell division upon extracellular stimulation and is sufficient to support tumourigenesis when permanently triggered by activating mutations. Surprisingly, even strong signalling protein overexpression usually does not trigger deregulated cell proliferation, suggesting that basal state signalling is insensitive to wildtype protein overexpression. Using kinetic modelling of the core Ras cycle, we show that basal RasGTP signalling can be insensitive to Ras overexpression and thus identify a possible tumour suppression mechanism. We further show how phenotypically silent overexpression events within signalling cascades cooperate to bring about carcinogenesis. Our analyses underscore the need for a systems level understanding of tumour formation.     

Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD₆₀₀ of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD₆₀₀ of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD₆₀₀ of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular.     

Molecular identification of a novel β-1,3-glucanase from alkaliphilic Nocardiopsis sp. strain F96

Alkaliphilic Nocardiopsis sp. strain F96 produced three -1,3-glucanase isozymes of different molecular masses (BglF1, BglF2 and BglF3). The N-terminal amino acid sequences of BglFs indicated that these isozymes were the products of a single gene. The -1,3-glucanase gene (bglF) was cloned from the chromosomal DNA of strain F96. The bglF gene encoded a polypeptide of 270 amino acids including a signal sequence. The deduced amino acid sequence of mature BglF exhibited the highest homology to those of glycoside hydrolase (GH) family 16 -1,3-glucanases, suggesting that the enzyme belonged to the GH family 16. The mature region of bglF gene was functionally expressed in Escherichia coli. The optimum pH and temperature of purified recombinant BglF were pH 9.0 and 70°C, respectively. This enzyme efficiently hydrolyzed insoluble -1,3-glucans and showed the highest activity toward a -1,3-1,4-glucan rather than -1,3-glucans. These results suggested that BglF would be a novel -1,3-glucanse. Mutational analysis revealed that Glu123 and Glu128 should be the catalytic residues of BglF.     

心肌特异性高表达热休克蛋白27转基因鼠建立

目的 建立人热休克蛋白27（heat shock protein 27, Hsp27）基因在小鼠心肌特异性表达的转基因鼠模型。 方法 将人心肌Hsp27cDNA插入含有心肌特异性表达启动子αMHC的pBSⅡ-SK+载体中,限制性内切酶EcoRI酶切、纯化后,获得含有αMHC启动子－Hsp27cDNA－HGH polyA的线性DNA片段,以显微注射法将目的基因导入受精卵, PCR筛选基因型,Western Blot鉴定转移基因的表达和表达的组织特异性。结果和结论 共获得两个转基因系小鼠,均呈心肌组织特异性高表达。     

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX₁ promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X₃₃ and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X₃₃ cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His₆-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His₆-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His₆-ROLw) and the mutant (His₆-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.     

The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His₆-tagged EcDCP (His₆-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni²⁺-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His₆-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His₆-EcDCP was stimulated by 1.5 fold. The K _M and k _cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s⁻¹, respectively. His₆-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His₆-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.     

Overexpression of <Emphasis Type="Italic">Citrus junos</Emphasis> mitochondrial citrate synthase gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> confers aluminum tolerance

Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.     

Optimized production and analysis of the staphylococcal multidrug efflux protein QacA

The plasmid-encoded QacA multidrug transport protein confers high-level resistance to a range of commonly used antimicrobials and is carried by widespread clinical strains of the human pathogen Staphylococcus aureus making it a potential target for future drug therapies. In order to obtain a sufficient yield of QacA protein for structural and biophysical studies, an optimized strategy for QacA overexpression was developed. QacA expression, directed from several vector systems in Escherichia coli, was tested under various growth and induction conditions and a synthetic qacA gene, codon-optimized for expression in E. coli was developed. Despite the extreme hydrophobicity and potential toxicity of the QacA secondary transport protein, a strategy based on the pBAD expression system, yielding up to four milligrams of approximately 95% pure QacA protein per litre of liquid culture, was devised. Purified QacA protein was examined using circular dichroism spectroscopy and displayed a secondary structure akin to that predicted from in silico analyses. Additionally, detergent solubilized QacA protein was shown to bind its fluorescent substrate rhodamine 6G with micro-molar affinity using a fluorescence polarization-based binding assay, similar to other multidrug transport proteins. To check the applicability of the expression/purification system described for QacA to other staphylococcal secondary transporters, the gene encoding the TetA(K) tetracycline efflux protein, which was previously recalcitrant to overexpression, was incorporated into the pBAD-based system and shown to be readily produced at easily detectable levels. Therefore, this expression system could be of general use for the production of secondary transport proteins in E. coli.