Desmin mutations impact the autophagy flux in C2C12 cell in mutation-specific manner.

Desmin is the main intermediate filament of striated and smooth muscle cells and plays a crucial role in maintaining the stability of muscle fiber during contraction and relaxation cycles. Being a component of Z-disk area, desmin integrates autophagic pathways, and the disturbance of Z-disk proteins' structure negatively affects chaperone-assisted selective autophagy (CASA). In the present study, we focused on alteration of autophagy flux in myoblasts expressing various Des mutations. We applied Western blotting, immunocytochemistry, RNA sequencing, and shRNA approach to demonstrate that DesS12F, DesA357P, DesL345P, DesL370P, and DesD399Y mutations. Mutation-specific effect on autophagy flux being most severe in aggregate-prone Des mutations such as DesL345P, DesL370P, and DesD399Y. RNA sequencing data confirmed the most prominent effect of these mutations on expression profile and, in particular, on autophagy-related genes. To verify CASA contribution to desmin aggregate formation, we suppressed CASA by knocking down Bag3 and demonstrated that it promoted aggregate formation and lead to downregulation of Vdac2 and Vps4a and upregulation of Lamp, Pink1, and Prkn. In conclusion, Des mutations showed a mutation-specific effect on autophagy flux in C2C12 cells with either a predominant impact on autophagosome maturation or on degradation and recycling processes. Aggregate-prone desmin mutations lead to the activation of basal autophagy level while suppressing the CASA pathway by knocking down Bag3 can promote desmin aggregate formation.

Monocytes with Oncogenic Mutation JAK2 V617F as a Tool for Studies of the Pathogenic Mechanisms of Myelofibrosis.

We analyzed previously generated stable monocyte-derived cell line carrying mutation JAK2 V617F. Evaluation of the expression of pro- and antifibrotic factors revealed changes in the production of MMPs and their inhibitors, growth factors, galectin-3, and pentraxin 3 in cells carrying mutation JAK2 in comparison with control non-modified cells.

[Murine and human hematopoietic progenitor cultures grown on stromal layers expressing Notch ligands].

The ex vivo maintenance and expansion of hematopoietic stem cells and early progenitors is necessary for the successful treatment of hematopoietic and immune diseases. Multiple attempts to improve the expansion of hematopoietic stem cells (HSCs) by their cultivation in the presence of growth factor cocktails have so far failed. Novel approaches aimed at conserving the earliest precursors in their undifferentiated state are needed. These approaches should take into account local regulatory factors that are present in the HSC microenvironment and the three-dimensional architecture of their niche. In the present study, we compared the effects of two Notch ligands, i.e., Jagged1 and DLL1, on murine and human hematopoiesis in vitro. Our observations indicate that the stromal expression of Notch ligands increases the production of both the total and phenotypically early murine and human hematopoietic cells in the co-culture. On one hand, this study demonstrates the similarity of effects of stromal expression of Notch ligands on murine and human hematopoiesis in vitro. On the other hand, our study revealed a number of cell type and ligand-specific variations that are systematically described below. It seems that the effects of SCF cytokine addition on murine hematopoiesis in vitro depend on the stromal context and are oppositely directed for Jagged1 and DLL1.

[The role of ZF5 and CGGBP-20 transcription factors in expression regulation of human FMR1 gene responsible for X-fragile syndrome].

The human FMR1 gene encodes an RNA-binding protein taking part in translation regulation. The 5'-untranslated region of FMR1 gene contains a large number of tandem repeats of GCC triplets (5-50) which increasing (more then 200) is responsible for X-fragile syndrome (human congenital anomaly). As it has been shown earlier, al least two transcription factors (ZF5 and CGGBP-20) are capable of interacting specifically with GCC-repeats in regulatory regions of some genes. In this work, their roles in FMR1 gene expression regulation were studied. It was demonstrated by electrophoretic mobility shift assay that ZF5 recombinant protein specifically bound with GCC-triplet repeats (GCC9). Tissue-specific distributions of ZF5 and FMR1 proteins are very overlapped in mammalian. Inhibition of ZF5 expression in HepG2 cells (by RNA interference) leads to at least 1.5 times stimulations of FMR1 gene expression in these cells. To estimate the contribution of GCC-triplet repeats in FMR1 gene expression regulation we used two alternative variants of genetic construction: containing luciferase reporter gene under 5'-regulatory region fragment devoid of GCC-triplet repeats or including the GCC9 nucleotide sequence. HepG2 cells were co-transfected by these constructions and expressions vectors of ZF5 or (and) CGGBP-20 respectively. It was found that ZF5 downregulated the activity of 5'-regulatory region of FMR1 gene in both cases (acting probably through canonic 5'-GCGCGC3' sites). The presence of GCC-triplet repeats in the construction weakens this ZF5 effect. CGGBP-20 downregulates the activity of 5'-region of FMR1 gene in the presence of GCC-triplets only. The data obtained evidently indicate differently directed ZF5 effects on FMR1 gene expression and suggest the mechanism to explain the earlier demonstrated phenomenon about increasing of mRNA level in permutation FMR1 allele carries.