A sterol-binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing.

Kes1, and other oxysterol-binding protein superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids and dampens gene expression driven by Gcn4, the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic "large Mediator" complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing.

Lidocaine promotes apoptosis in breast cancer cells by affecting VDAC1 expression.

OBJECTIVE: To investigate the effect of lidocaine on the expression of voltage-dependent anion channel 1 (VDAC1) in breast invasive carcinoma (BRCA) and its impact on the apoptosis of breast cancer cells. METHODS: We collected clinical data from patients with invasive breast cancer from 2010 to 2020 in the First affiliated hospital of Nanchang University, evaluated the prognostic value of VDAC1 gene expression in breast cancer, and detected the expression of VDAC1 protein in breast cancer tissues and paracancerous tissues by immunohistochemical staining of paraffin sections. Also, we cultured breast cancer cells (MCF-7) to observe the effect of lidocaine on the apoptosis of MCF-7 cells. RESULTS: Analysis of clinical data and gene expression data of BRCA patients showed VDAC1 was a differentially expressed gene in BRCA, VDAC1 may be of great significance for the diagnosis and prognosis of BRCA patients. Administration of lidocaine 3 mM significantly decreased VDAC1 expression, the expression of protein Bcl-2 was significantly decreased (p < 0.05), and the expression of p53 increased significantly (p < 0.05). Lidocaine inhibited the proliferation of MCF-7 breast cancer cells, increased the percentage of G2 / M phase cells and apoptosis. CONCLUSION: Lidocaine may inhibit the activity of breast cancer cells by inhibiting the expression of VDAC1, increasing the apoptosis in breast cancer cells.

Helicobacter pylori infection promotes epithelial-to-mesenchymal transition of gastric cells by upregulating LAPTM4B.

Helicobacter pylori infection can lead to epithelial-to-mesenchymal transition (EMT) and the progression of gastric cancer (GC); however, the underlying mechanism is poorly understood. Lysosomal-associated protein transmembrane 4ß (LAPTM4B) has been implicated in carcinogenesis, including in GC, and we previously showed that LAPTM4B-35 overexpression was an independent prognostic factor in GC. In this study, we demonstrate that upregulation of LAPTM4B promotes GES-1 human gastric epithelial cell proliferation, migration, and invasion and EMT. Conversely, LAPTM4B downregulation inhibited proliferation, migration, invasion, and EMT in SGC7901 GC cells. We also found that H. pylori infection enhanced LAPTM4B expression and induced EMT in GES-1 cells. Thus, EMT in GC is promoted by a combination of LAPTM4B overexpression and H. pylori infection. These results provide a basis for the development of novel two-pronged therapeutic strategies for the treatment of GC.

PITPs as targets for selectively interfering with phosphoinositide signaling in cells.

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs nor PITPs in general have been exploited as targets for chemical inhibition for such purposes. Herein, we validate what is to our knowledge the first small-molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs) and are effective inhibitors in vitro and in vivo. We further establish that Sec14 is the sole essential NPPM target in yeast and that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects and demonstrate that NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof of concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies.

Noncanonical regulation of phosphatidylserine metabolism by a Sec14-like protein and a lipid kinase.

The yeast phosphatidylserine (PtdSer) decarboxylase Psd2 is proposed to engage in a membrane contact site (MCS) for PtdSer decarboxylation to phosphatidylethanolamine (PtdEtn). This proposed MCS harbors Psd2, the Sec14-like phosphatidylinositol transfer protein (PITP) Sfh4, the Stt4 phosphatidylinositol (PtdIns) 4-OH kinase, the Scs2 tether, and an uncharacterized protein. We report that, of these components, only Sfh4 and Stt4 regulate Psd2 activity in vivo. They do so via distinct mechanisms. Sfh4 operates via a mechanism for which its PtdIns-transfer activity is dispensable but requires an Sfh4-Psd2 physical interaction. The other requires Stt4-mediated production of PtdIns-4-phosphate (PtdIns4P), where Stt4 (along with the Sac1 PtdIns4P phosphatase and endoplasmic reticulum-plasma membrane tethers) indirectly modulate Psd2 activity via a PtdIns4P homeostatic mechanism that influences PtdSer accessibility to Psd2. These results identify an example in which the biological function of a Sec14-like PITP is cleanly uncoupled from its canonical in vitro PtdIns-transfer activity and challenge popular functional assumptions regarding lipid-transfer protein involvements in MCS function.

Structure and function of the Saccharomyces cerevisiae Sir3 BAH domain.

Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homology) domain at its N terminus. Overexpression of this domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH domains. The structure of the Sir3 BAH domain has been determined. In the crystal, the molecule multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH domain of Sir3 in silencing.

[Expression, refolding and biological activity of recombinant type-I metalloproteinase acutolysin a from Agkistrodon acutus].

Type-I snake venom metalloproteinase acutolysin A gene was cloned into the prokaryotic expression vector pBAD/gIIIA and the resulting recombinant plasmid pDS was obtained. By the induction with 0.02% L-(+)-arabinose, the recombinant metalloproteinase was expressed in insoluble inclusion body in E. coli TOP10 and reached up to 5%--10% of total bacterial proteins. The recombinant metalloproteinase has an additional sequence of N-terminal 22 amino acids and C-terminal 8 amino acids (housing 6 histidines), both of which derived from the vector. The purified inclusion body was solubilized by 8 mol/L urea or 6 mol/L guanidine-HCl and the denatured soluble recombinant metalloproteinase was allowed to refold in vitro. Western blotting and ELISA obviously showed that the renatured recombinant metalloproteinase possessed strong immune reactivity very closely related to natural acutolysin A. Animal experiments showed that the refolded recombinant metalloproteinase had an obvious hemorrhagic activity. Except PMSF, 1 mmol/L EDTA, 1 mmol/L EGTA, and 3 mmol/L imidazole could inhibit the hemorrhagic activity of the recombinant and the natural metalloproteinases to different extent. Based on the investigations of others and our experimental results, the hemorrhagic mechanism of snake metalloproteinases was discussed.

Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution.

Sec14-superfamily proteins integrate the lipid metabolome with phosphoinositide synthesis and signaling via primed presentation of phosphatidylinositol (PtdIns) to PtdIns kinases. Sec14 action as a PtdIns-presentation scaffold requires heterotypic exchange of phosphatidylcholine (PtdCho) for PtdIns, or vice versa, in a poorly understood progression of regulated conformational transitions. We identify mutations that confer Sec14-like activities to a functionally inert pseudo-Sec14 (Sfh1), which seemingly conserves all of the structural requirements for Sec14 function. Unexpectedly, the "activation" phenotype results from alteration of residues conserved between Sfh1 and Sec14. Using biochemical and biophysical, structural, and computational approaches, we find the activation mechanism reconfigures atomic interactions between amino acid side chains and internal water in an unusual hydrophilic microenvironment within the hydrophobic Sfh1 ligand-binding cavity. These altered dynamics reconstitute a functional "gating module" that propagates conformational energy from within the hydrophobic pocket to the helical unit that gates pocket access. The net effect is enhanced rates of phospholipid-cycling into and out of the Sfh1* hydrophobic pocket. Taken together, the directed evolution approach reveals an unexpectedly flexible functional engineering of a Sec14-like PtdIns transfer protein-an engineering invisible to standard bioinformatic, crystallographic, and rational mutagenesis approaches.

Mutational analysis of the Sir3 BAH domain reveals multiple points of interaction with nucleosomes.

Sir3, a component of the transcriptional silencing complex in the yeast Saccharomyces cerevisiae, has an N-terminal BAH domain that is crucial for the protein's silencing function. Previous work has shown that the N-terminal alanine residue of Sir3 (Ala2) and its acetylation play an important role in silencing. Here we show that the silencing defects of Sir3 Ala2 mutants can be suppressed by mutations in histones H3 and H4, specifically, by H3 D77N and H4 H75Y mutations. Additionally, a mutational analysis demonstrates that three separate regions of the Sir3 BAH domain are important for its role in silencing. Many of these BAH mutations also can be suppressed by the H3 D77N and H4 H75Y mutations. In agreement with the results of others, in vitro experiments show that the Sir3 BAH domain can interact with partially purified nucleosomes. The silencing-defective BAH mutants are defective for this interaction. These results, together with the previously characterized interaction between the C-terminal region of Sir3 and the histone H3/H4 tails, suggest that Sir3 utilizes multiple domains to interact with nucleosomes.