Effects of his-tags on physical properties of parvalbumins

A comparative study of His-tagged and non-tagged rat β-parvalbumin (rWT β-PA), calcium binding protein with the EF-hand calcium binding domains, has been carried out. The attachment of His-tag increases α-helical content and decreases β-sheets and β-turns content of the metal free form (apo-state) of β-PA. In contrast to this, the attachment of His-tag decreases α-helical content by more than 10% and increases contents of β-sheets and β-turns of the Ca²⁺-loaded state. According to the dynamic light scattering analysis, apo-state of His-tagged rat β-PA seems to be less compact compared with the apo-state of non-tagged rat β-PA. Surprisingly, the attachment of His-tag practically does not change mean hydrodynamic radius of Ca²⁺-loaded rat β-PA. The attachment of His-tag shifts thermal denaturation peaks of both apo- and Ca²⁺-loaded states of rat β-PA towards higher temperatures by 3–4 °C and slightly decreases its Ca²⁺ affinity. These results should be taken into consideration in the use of His-tagged parvalbumins.     

Microbially Induced Calcite Precipitation for Seepage Control in Sandy Soil

Microbially induced calcite precipitation (MICP) can reduce the permeability of soil by reducing the pore volumes. A MICP-based soil improvement method to control water leakage in irrigation channels and reservoirs built on sandy soil grounds is presented in this article. Using this method, a low-permeable hard crust can be formed at the soil surfaces. An experimental study was carried out to evaluate the effect of this method. Sandy soil samples were treated using four different schemes, namely, (1) surface spray, (2) surface spray with the addition of fibers, (3) surface spray and bulk stabilization, and (4) immersion stabilization. By applying around 2.6?L treatment liquid (consisting of ureolytic bacteria, 0.5?mol/L calcium chloride and 0.5?mol/L urea) to the top 2-cm thick soil, the seepage rates of the samples treated by the four different schemes could be reduced by up to 379 times. The conversion rates of calcium source in the tests were up to 89.7%. The results showed that a method of treating the soil in bulk before the formation of a crust on top of the soil layer was effective in reducing the seepage rates. After the bio-treatment, the formed low-permeable hard crust layer was 10 to 20?mm thick with a calcite content higher than 5%. Below the hard crusts, the calcite content was less than 5% and the soil was not properly cemented. Using the mercury intrusion test, it was found that both pore volumes and pore sizes of the bio-treated soil reduced significantly as compared with the untreated soil. Penetration tests using a flat-bottom penetrometer were used to assess the mechanical behavior of the bio-treated soil. The results indicated that the penetration resistance of the bio-treated soil layer was much higher than that of the untreated soil.     

Comparison of microbially induced calcium carbonate precipitation eligibility using sporosarcina pasteurii and bacillus licheniformis on two different sands

The use of biological means for ground improvement have become popular, which generally works through the process called microbially-induced calcium carbonate precipitation (MICP). Many studies indicate successful application of MICP based improvement with multiple bacteria and on several soils. Given the proven performance of MICP, this study aims to examine the MICP process by comparing the calcium carbonate precipitation ability of widely studied bacteria, i.e., Sporosarcina pasteurii and relatively under-recognized bacteria, i.e., Bacillus licheniformis to outline the formation success. For this purpose, two different sands were tested for observing precipitation behavior using a series of syringe tests. Furthermore, the effect of concentration and inclusion of calcium chloride for nutrition of bacteria, saturation with water, and hybrid use of two bacteria were investigated in some tests for diversification. X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive x-ray spectroscopy (EDS) were used for the interpretation of results. Results indicated that Sporosarcina pasteurii had performed superior over Bacillus licheniformis when achieving calcium carbonate precipitation in tests for both sands. In addition, many intriguing SEM images contributed to the literature of MICP monitoring, highlighting the effects of the variables investigated.     

Characterization of an RNase with two catalytic centers. Human RNase6 catalytic and phosphate-binding site arrangement favors the endonuclease cleavage of polymeric substrates

Background

Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site.

A novel method for cloning of coding sequences of highly toxic proteins

Background

During standard gene cloning, the recombinant protein appearing in bacteria as the result of expression leakage very often inhibits cell proliferation leading to blocking of the cloning procedure. Although different approaches can reduce transgene basal expression, the recombinant proteins, which even in trace amounts inhibit bacterial growth, can completely prevent the cloning process.

Sequence- and seed-structure-dependent polymorphic fibrils of alpha-synuclein

Synucleinopathies comprise a diverse group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. These share a common pathological feature, the deposition of alpha-synuclein (a-syn) in neurons or oligodendroglia. A-syn is highly conserved in vertebrates, but the primary sequence of mouse a-syn differs from that of human at seven positions. However, structural differences of their aggregates remain to be fully characterized. In this study, we found that human and mouse a-syn aggregated in vitro formed morphologically distinct amyloid fibrils exhibiting twisted and straight structures, respectively. Furthermore, we identified different protease-resistant core regions, long and short, in human and mouse a-syn aggregates. Interestingly, among the seven unconserved amino acids, only A53T substitution, one of the familial PD mutations, was responsible for structural conversion to the straight-type. Finally, we checked whether the structural differences are transmissible by seeding and found that human a-syn seeded with A53T aggregates formed straight-type fibrils with short protease-resistant cores. These results suggest that a-syn aggregates form sequence-dependent polymorphic fibrils upon spontaneous aggregation but become seed structure-dependent upon seeding.     

Phosphorylation of NHERF1 S279 and S301 differentially regulates breast cancer cell phenotype and metastatic organotropism

Metastatic cancer cells are highly plastic for the expression of different tumor phenotype hallmarks and organotropism. This plasticity is highly regulated but the dynamics of the signaling processes orchestrating the shift from one cell phenotype and metastatic organ pattern to another are still largely unknown. The scaffolding protein NHERF1 has been shown to regulate the expression of different neoplastic phenotypes through its PDZ domains, which forms the mechanistic basis for metastatic organotropism. This reprogramming activity was postulated to be dependent on its differential phosphorylation patterns. Here, we show that NHERF1 phosphorylation on S279/S301 dictates several tumor phenotypes such as in vivo invasion, NHE1-mediated matrix digestion, growth and vasculogenic mimicry. Remarkably, injecting mice with cells having differential NHERF1 expression and phosphorylation drove a shift from the predominantly lung colonization (WT NHERF1) to predominately bone colonization (double S279A/S301A mutant), indicating that NHERF1 phosphorylation also acts as a signaling switch in metastatic organotropism.     

Nod1-mediated lipolysis promotes diacylglycerol accumulation and successive inflammation via PKCδ-IRAK axis in adipocytes

Chronic inflammation contributes to obesity mediated metabolic disturbances, including insulin resistance. Obesity is associated with altered microbial load in metabolic tissues that can contribute to metabolic inflammation. Different bacterial components such as, LPS, peptidoglycans have been shown to underpin metabolic disturbances through interaction with host innate immune receptors. Activation of Nucleotide-binding oligomerization domain-containing protein 1 (Nod1) with specific peptidoglycan moieties promotes insulin resistance, inflammation and lipolysis in adipocytes. However, it was not clear how Nod1-mediated lipolysis and inflammation is linked. Here, we tested if Nod1-mediated lipolysis caused accumulation of lipid intermediates and promoted cell autonomous inflammation in adipocytes. We showed that Nod1-mediated lipolysis caused accumulation of diacylglycerol (DAG) and activation of PKCδ in 3T3-L1 adipocytes, which was prevented with a Nod1 inhibitor. Nod1-activated PKCδ caused downstream stimulation of IRAK1/4 and was associated with increased expression of proinflammatory cytokines such as, IL-1β, IL-18, IL-6, TNFα and MCP-1. Pharmacological inhibition or siRNA mediated knockdown of IRAK1/4 attenuated Nod1-mediated activation of NF-κB, JNK, and the expression of proinflammatory cytokines. These results reveal that Nod1-mediated lipolysis promoted accumulation of DAG, which engaged PKCδ and IRAK1/4 to augment inflammation in 3T3-L1 adipocytes.     

Glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: Strategies of protein stabilization

Abstract: The molecular origin of protein stability has been the subject of active research for more than a generation (R. Jaenicke (1991) Eur. J. Biochem. 202, 715–728). Faced with the discovery of extremophiles, in recent years the problem has gained momentum, especially because of its biotechnological potential. In analyzing a number of enzymes from the hyperthermophilic bacterium Thermotoga maritima , it has become clear that the excess free energy of stabilization is equivalent to only a few weak bonds ( ΔΔG _stab≈ 50 kJ/mol). As taken from the comparison of homologous enzymes from mesophiles, thermophiles and hyperthermophiles, these accumulate from local interactions (especially ion pairs), enhanced secondary or supersecondary structure, and improved packing of domains and/or subunits, without significantly altering the overall topology. In this review, glyceraldehyde-3-phosphate dehydrogenase will be discussed as a representative example to illustrate possible adaptive strategies to the extreme thermal stress in hydrothermal vents.