microRNA-15b contributes to depression-like behavior in mice by affecting synaptic protein levels and function in the nucleus accumbens.

Major depression is a prevalent affective disorder characterized by recurrent low mood. It presumably results from stress-induced deteriorations of molecular networks and synaptic functions in brain reward circuits of genetically-susceptible individuals through epigenetic processes. Epigenetic regulator microRNA-15b inhibits neuronal progenitor proliferation and is up-regulated in the medial prefrontal cortex of mice that demonstrate depression-like behavior, indicating the contribution of microRNA-15 to major depression. Using a mouse model of major depression induced by chronic unpredictable mild stress (CUMS), here we examined the effects of microRNA-15b on synapses and synaptic proteins in the nucleus accumbens of these mice. The application of a microRNA-15b antagomir into the nucleus accumbens significantly reduced the incidence of CUMS-induced depression and reversed the attenuations of excitatory synapse and syntaxin-binding protein 3 (STXBP3A)/vesicle-associated protein 1 (VAMP1) expression. In contrast, the injection of a microRNA-15b analog into the nucleus accumbens induced depression-like behavior as well as attenuated excitatory synapses and STXBP3A/VAMP1 expression similar to the down-regulation of these processes induced by the CUMS. We conclude that microRNA-15b-5p may play a critical role in chronic stress-induced depression by decreasing synaptic proteins, innervations, and activities in the nucleus accumbens. We propose that the treatment of anti-microRNA-15b-5p may convert stress-induced depression into resilience.

Intrinsic defects in erythroid cells from familial hemophagocytic lymphohistiocytosis type 5 patients identify a role for STXBP2/Munc18-2 in erythropoiesis and phospholipid scrambling.

Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is a rare genetic disorder caused by mutations in STXBP2/Munc18-2. Munc18-2 plays a role in the degranulation machinery of natural killer cells and cytotoxic T lymphocytes. Mutations in STXBP2/Munc18-2 lead to impaired killing of target cells by natural killer cells and cytotoxic T lymphocytes, which in turn results in elevated levels of the inflammatory cytokine interferon γ, macrophage activation, and hemophagocytosis. Even though patients with FHL-5 present with anemia and hemolysis, no link between the disease and the erythroid lineage has been established. Here we report that red blood cells express Munc18-2 and that erythroid cells from patients with FHL-5 exhibit intrinsic defects caused by STXBP2/Munc18-2 mutations. Red blood cells from patients with FHL-5 expose less phosphatidylserine on their surface upon Ca(2+) ionophore ionomycin treatment. Furthermore, cultured erythroblasts from patients with FHL-5 display defective erythropoiesis characterized by decreased CD235a expression and aberrant cell morphology.

Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.

Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1-2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.

Effect of thyroxine on munc-18 and syntaxin-1 expression in dorsal hippocampus of adult-onset hypothyroid rats.

Adult-onset hypothyroidism induces a variety of impairments on hippocampus-dependent neurocognitive functioningin which many synaptic proteins in hippocampus neurons are involved. Here, we observed the effect of adult-onset hypothyroidism on the expression of syntaxin-1 and munc-18 in the dorsal hippocampus and whether the altered proteins could be restored by levothyroxine (T4) treatment. All rats were separated into 4 groups randomly: hypothyroid group, 5 µg T4/100 g body weight (BW) treated group, 20 µg T4/100g BW treated group and control group. The radioimmunoassay kits were applied to assay the levels of serum T3 and T4, and the levels of syntaxin-1 and munc-18 in hippocampus were assessed by immunohistochemistry and Western blot. Both analysis corroborated that syntaxin-1 in the hypothyroid group was significantly higher. Munc-18 was lower in four layers of CA3 and dentate gyrus by immunohistochemistry. After two weeks of treatment with 5 µg T4/100g BW for hypothyroidism, syntaxin-1 levels were completely restored, whereas the recovery of munc-18 only located in two of the four impaired layers. Twenty µg T4/100g BW treatment normalized munc-18 levels. These data suggested that adult-onset hypothyroidism induced increment of syntaxin-1 and decrement of munc-18 in the dorsal hippocampus, which could be restored by T4 treatment. Larger dosage of T4 caused more effective restorations.

Protein expression of PKCZ (Protein Kinase C Zeta), Munc18c, and Syntaxin-4 in the insulin pathway in endometria of patients with polycystic ovary syndrome (PCOS).

BACKGROUND: Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic disorder commonly associated with insulin resistance (IR). Previous studies indicate about the expression of molecules involved in the insulin pathway in endometria of women with PCOS-IR. Therefore, the aim of the present study was to evaluate the effect of insulin and testosterone in the expression of these proteins in the endometria and immortal endometrial stromal cell line (T-HESCs). METHODS: We examined the protein levels of Munc18c, PKC zeta, phospho-PKC Zeta, and Syntaxin-4. Protein levels were assessed by Western Blot and/or immunohistochemistry in proliferative endometria (NPE = 6) and in PCOS endometria with insulin resistance (PCOSE-IR = 6). We also evaluated whether high concentrations of insulin (100 nM) and/or testosterone (100 nM), during a 24 h stimulatory period, affected the expression of these proteins in an immortal endometrial stromal cell line (T-HESCs). Once stimulated, proteins were extracted from cells and were assessed by Western Blot analysis. Immunocytochemistry was performed to detect AR in T-HESC cells. RESULTS: Western Blot data showed decreased expression (p < 0,05) of Munc18c and phospho-PKC Zeta in PCOS-IR endometria (PCOSE-IR) with respect to the control (NPE). In the in vitro study, Western Blot analysis showed decreased levels of Munc18c, PKC Zeta and phospho-PKC Zeta with the different hormonal treatments when compared to the control condition (no hormonal stimulation) (p < 0,05). The AR was present in the endometrial stromal cell line (T-HESC). CONCLUSION: The conditions of hyperinsulinism and hyperandrogenism present in PCOS-IR patients modulate the expression and/or phosphorylation of the proteins involved in the insulin pathway at the endometrial level. These data extend to the T-HESCs cells results, where insulin and testosterone exert an effect on both the expression and phosphorylation of proteins present in the pathway.

Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and α-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae.

The vesicle-trafficking protein munc18b increases the secretory capacity of mammalian cells.

Heterologous protein production in mammalian cells is often challenged by the bottleneck of the secretory machinery, which prevents producer cells from fully exploiting their physiologic capacity in the production of biopharmaceuticals. Recent advances in the understanding of the molecular mechanisms of vesicle trafficking have enabled the identification of key regulators that control the flow of recombinant proteins along the secretory pathway. Here, we report that transgenic expression of Munc18b, a Sec1/Munc18 (SM) protein regulating the fusion of secretory vesicles to the plasma membrane, enhances the secretory capacity of HeLa, HEK-293 and HT-1080 and so increases overall production of different secreted human glycoproteins as well as the titer of lentiviral particles produced in HEK-293-derived helper cells. Targeted interventions in secretory vesicle trafficking by Munc18b is a novel secretion engineering strategy, which harnesses the full secretory capacity of mammalian cells. Secretion engineering is the latest-generation metabolic engineering strategy, which could improve future therapies by increasing the production of biopharmaceuticals by boosting the secretion performance of cell implants in cell therapy initiatives and by raising the production titers of transgenic viral particles used for gene therapy applications.

Distinct modulation of Kv1.2 channel gating by wild type, but not open form, of syntaxin-1A.

SNARE proteins, syntaxin-1A (Syn-1A) and SNAP-25, inhibit delayed rectifier K(+) channels, K(v)1.1 and K(v)2.1, in secretory cells. We showed previously that the mutant open conformation of Syn-1A (Syn-1A L165A/E166A) inhibits K(v)2.1 channels more optimally than wild-type Syn-1A. In this report we examined whether Syn-1A in its wild-type and open conformations would exhibit similar differential actions on the gating of K(v)1.2, a major delayed rectifier K(+) channel in nonsecretory smooth muscle cells and some neuronal tissues. In coexpression and acute dialysis studies, wild-type Syn-1A inhibited K(v)1.2 current magnitude. Of interest, wild-type Syn-1A caused a right shift in the activation curves of K(v)1.2 without affecting its steady-state availability, an inhibition profile opposite to its effects on K(v)2.1 (steady-state availability reduction without changes in voltage dependence of activation). Also, although both wild-type and open-form Syn-1A bound equally well to K(v)1.2 in an expression system, open-form Syn-1A failed to reduce K(v)1.2 current magnitude or affect its gating. This is in contrast to the reported more potent effect of open-form Syn-1A on K(v)2.1 channels in secretory cells. This finding together with the absence of Munc18 and/or 13-1 in smooth muscles suggested that a change to an open conformation Syn-1A, normally facilitated by Munc18/13-1, is not required in nonsecretory smooth muscle cells. Taken together with previous reports, our results demonstrate the multiplicity of gating inhibition of different K(v) channels by Syn-1A and is compatible with versatility of Syn-1A modulation of repolarization in various secretory and nonsecretory (smooth muscle) cell types.