[The Titer of the Lentiviral Vector Encoding Chimeric TRIM5α-HRH Gene is Reduced Due to Expression of TRIM5α-HRH in Producer Cells and the Negative Effect of Efla Promoter].

The chimeric protein TRIM5α-HRH is a promising antiviral factor for HIV-1 gene therapy. This protein is able to protect cells from HIV-1 by blocking the virus in the cytoplasm. We are developing protocol of HIV-1 gene therapy, which involves the delivery of the TRIM5α-HRH gene into CD4^(+) T-lymphocytes by lentiviral vectors (LVs). However, LVs containing TRIM5α-HRH have a low infectious titer, which prevents effective T cell modification. Here, we found that the expression of TRIM5α-HRH during pseudoviral particle production in HEK293 T cells, as well as the presence of the Eflα promoter in our construction are responsible for titer reduction. These results allow us to determine the directions for further optimization of LV with the TRIM5α-HRH gene to improve its infectious titer.

α-Synuclein Overexpression in SH-SY5Y Human Neuroblastoma Cells Leads to the Accumulation of Thioflavin S-positive Aggregates and Impairment of Glycolysis.

Deterioration of energy metabolism in affected cells is an important feature of synucleinopathies, including Parkinson's disease. Here, we studied the association between α-synuclein accumulation and glycolysis using SH-SY5Y neuroblastoma cell lines stably expressing wild-type α-synuclein or its A53T mutant linked to the autosomal dominant form of the disease. Overexpression of both proteins led to the accumulation of thioflavin S-positive aggregates, more pronounced for α-synuclein A53T. It also caused changes in the cell energy metabolism manifested as a decrease in the lactate accumulation and glucose uptake. Impairments in glycolysis were also accompanied by a decrease in the activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In vitro experiments with purified proteins indicated that GAPDH inactivation might be caused by its binding to the monomeric and oligomeric forms of α-synuclein. Therefore, a decrease in the GAPDH activity induced by its interaction with α-synuclein, might be one of the causes of glucose metabolism deterioration in synucleinopathies.

Pectin methylesterase-generated methanol may be involved in tobacco leaf growth.

Plant leaves undergo a sink-source modification of intercellular macromolecular transport during the transition from carbon import to carbon export. After assessing the role of metabolite signaling in gene regulation in Nicotiana tabacum sink and source leaves, we observed increased pectin methylesterase (PME)-mediated methanol generation in immature leaves. Using suppression subtractive hybridization (SSH), we identified a number of genes whose activity changes from sink to source leaves. The most abundant SSH-identified genes appeared to be sensitive to methanol. We hypothesize that tobacco leaf maturation and the sink-source transition are accompanied by a change in mRNA levels of genes that function in methanol-dependent cell signaling.

Rapid and massive green fluorescent protein production leads to formation of protein Y-bodies in plant cells.

Although high level of recombinant protein production can be achieved via transient expression in plant cells, the mechanism by which tolerance to the presence of recombinant protein is acquired remains unclear. Here we show that green fluorescent protein (GFP) encoded by an intron-optimized tobacco mosaic viral vector formed large membraneless GFP bodies called Y-bodies that demonstrated mainly perinuclear localization. The Y-bodies were heterogeneous in size, approaching the size of the cell nucleus. Experiments with extracted GFP and live cell imaging showed that Y-bodies included actively fluorescent, non-aggregated, tightly packed GFP molecules. The plant cells probably formed Y-bodies to exclude the recombinant protein from normal physiological turnover.