Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.     

Sex hormone modulation of cell growth and apoptosis of the human monocytic/macrophage cell line

Sex hormones seem to modulate the immune/inflammatory responses by different mechanisms in female and male rheumatoid arthritis patients. The effects of 17β-oestradiol and of testosterone were tested on the cultured human monocytic/macrophage cell line (THP-1) activated with IFN-γ in order to investigate their role in cell proliferation and apoptosis. Activated human THP-1 cells were cultured in the presence of 17β-oestradiol and testosterone (final concentration, 10 nM). The evaluation of markers of cell proliferation included the NF-κB DNA-binding assay, the NF-κB inhibition complex, the proliferating cell nuclear antigen expression and the methyl-tetrazolium salt test. Apoptosis was detected by the annexin V-propidium assay and by the cleaved poly-ADP ribose polymerase expression. Specific methods included flow analysis cytometry scatter analysis, immunocytochemistry and western blot analysis. Cell growth inhibition and increased apoptosis were observed in testosterone-treated THP-1 cells. Increased poly-ADP ribose polymerase-cleaved expression and decreased proliferating cell nuclear antigen expression, as well as an increase of IκB-α and a decrease of the IκB-α phosphorylated form (ser 32), were found in testosterone-treated THP-1 cells. However, the NF-κB DNA binding was found increased in 17β-oestradiol-treated THP-1 cells. The treatment with staurosporine (enhancer of apoptosis) induced decreased NF-κB DNA binding in all conditions, but particularly in testosterone-treated THP-1 cells. Treatment of THP-1 by sex hormones was found to influence cell proliferation and apoptosis. Androgens were found to increase the apoptosis, and oestrogens showed a protective trend on cell death – both acting as modulators of the NF-κB complex.     

Drastic changes in aquatic bacterial populations from the Cuatro Cienegas Basin (Mexico) in response to long-term environmental stress

Understanding the changes of aquatic microbial community composition in response to changes in temperature and ultraviolet irradiation is relevant for predicting biogeochemical modifications in the functioning of natural microbial communities under global climate change scenarios. Herein we investigate shifts in the bacterioplankton composition in response to long-term changes in temperature and UV radiation. For this purpose, 15 mesocosms were seeded with composite aquatic microbial communities from natural pools within the Cuatro Cienegas Basin (Mexican Chihuahuan desert) and were subject to different temperatures and UV conditions. 16S rRNA gene clone libraries were obtained from water samples at the mid-point (4 months) and the end of the experiment (8 months). An increase in bacterial diversity over time was found in the treatment of constant temperature and UV protection, which suggests that stable environments promote the establishment of complex and diverse bacterial community. Drastic changes in the phylogenetic bacterioplankton composition and structure were observed in response to fluctuating temperature and increasing UV radiation and temperature. Fluctuating temperature induced the largest decrease of bacterial richness during the experiment, indicating that frequent temperature changes drive the reduction in abundance of several species, most notably autotrophs. The long-term impact of these environmental stresses reduced diversity and selected for generalist aquatic bacterial populations, such as Porphyrobacter. These changes at the community level occur at an ecological time scale, suggesting that under global warming scenarios cascade effects on the food web are possible if the microbial diversity is modified.     

The PPARα agonist fenofibrate suppresses B-cell lymphoma in mice by modulating lipid metabolism

Obesity is associated with an increased risk for malignant lymphoma development. We used Bcr/Abl transformed B cells to determine the impact of aggressive lymphoma formation on systemic lipid mobilization and turnover. In wild-type mice, tumor size significantly correlated with depletion of white adipose tissues (WAT), resulting in increased serum free fatty acid (FFA) concentrations which promote B-cell proliferation in vitro. Moreover, B-cell tumor development induced hepatic lipid accumulation due to enhanced hepatic fatty acid (FA) uptake and impaired FA oxidation. Serum triglyceride, FFA, phospholipid and cholesterol levels were significantly elevated. Consistently, serum VLDL/LDL-cholesterol and apolipoprotein B levels were drastically increased. These findings suggest that B-cell tumors trigger systemic lipid mobilization from WAT to the liver and increase VLDL/LDL release from the liver to promote tumor growth. Further support for this concept stems from experiments where we used the peroxisome proliferator-activated receptor α (PPARα) agonist and lipid-lowering drug fenofibrate that significantly suppressed tumor growth independent of angiogenesis and inflammation. In addition to WAT depletion, fenofibrate further stimulated FFA uptake by the liver and restored hepatic FA oxidation capacity, thereby accelerating the clearance of lipids released from WAT. Furthermore, fenofibrate blocked hepatic lipid release induced by the tumors. In contrast, lipid utilization in the tumor tissue itself was not increased by fenofibrate which correlates with extremely low expression levels of PPARα in B-cells. Our data show that fenofibrate associated effects on hepatic lipid metabolism and deprivation of serum lipids are capable to suppress B-cell lymphoma growth which may direct novel treatment strategies. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

α-synuclein levels affect autophagosome numbers in vivo and modulate Huntington disease pathology

Huntington and Parkinson diseases (HD and PD) are two major neurodegenerative disorders pathologically characterized by the accumulation of the aggregate-prone proteins mutant huntingtin (in HD) and α-synuclein (in PD). Mutant huntingtin is an autophagy substrate and autophagy modulators affect HD pathology both in vitro and in vivo. In vitro, α-synuclein levels are able to modulate autophagy: α-synuclein overexpression inhibits autophagy, whereas downregulation promotes autophagy. Here, we review our recent studies showing that α-synuclein levels modulate mutant huntingtin toxicity in mouse models. This phenotypic modification is accompanied by the in vivo modulation of autophagosome numbers in mouse brains from both control and HD mice expressing different levels of α-synuclein.