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Listeria monocytogenes is a Gram-positive foodborne pathogen with high lethality and strong stress resistance, and its strong acid tolerance leads to many foodborne illnesses occurring in low-pH foods. Lactic acid is a generally recognized as safe (GRAS) food additive approved for use by the FDA.

Ubiquitination regulates immune signaling, and multiple E3 ubiquitin ligases have been studied in the context of their role in immunity. Despite this progress, the physiological roles of the Pellino E3 ubiquitin ligases, especially Pellino2, in immune regulation remain largely unknown. Accordingly, this study aimed to elucidate the role of Pellino2 in murine dendritic cells (DCs). In this study, we reveal a critical role of Pellino2 in regulation of the proinflammatory response following TLR9 stimulation. Pellino2-deficient murine DCs show impaired secretion of IL-6 and IL-12. Loss of Pellino2 does not affect TLR9-induced activation of NF-κB or MAPKs, pathways that drive expression of IL-6 and IL-12. Furthermore, DCs from Pellino2-deficient mice show impaired production of type I IFN following endosomal TLR9 activation, and it partly mediates a feed-forward loop of IFN-β that promotes IL-12 production in DCs. We also observe that Pellino2 in murine DCs is downregulated following TLR9 stimulation, and its overexpression induces upregulation of both IFN-β and IL-12, demonstrating the sufficiency of Pellino2 in driving these responses. This suggests that Pellino2 is critical for executing TLR9 signaling, with its expression being tightly regulated to prevent excessive inflammatory response. Overall, this study highlights a (to our knowledge) novel role for Pellino2 in regulating DC functions and further supports important roles for Pellino proteins in mediating and controlling immunity., Key Points Pellino2 mediates TLR9-induced cytokine production in dendritic cells.Pellino2 does not play a role in TLR9 signaling in macrophages.Pellino2 is a limiting factor for TLR9 signaling in dendritic cells.

Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is a protein with roles in early development, activation of the transcription factor NF-κB, and production of mitochondrial reactive oxygen species (mROS) that facilitates clearance of intracellular bacteria like Salmonella. ECSIT is also an important assembly factor for mitochondrial complex I. Unlike the murine form of Ecsit (mEcsit), we demonstrate here that human ECSIT (hECSIT) is highly labile. To explore whether the instability of hECSIT affects functions previously ascribed to its murine counterpart, we created a potentially novel transgenic mouse in which the murine Ecsit gene is replaced by the human ECSIT gene. The humanized mouse has low levels of hECSIT protein, in keeping with its intrinsic instability. Whereas low-level expression of hECSIT was capable of fully compensating for mEcsit in its roles in early development and activation of the NF-κB pathway, macrophages from humanized mice showed impaired clearance of Salmonella that was associated with reduced production of mROS. Notably, severe cardiac hypertrophy was manifested in aging humanized mice, leading to premature death. The cellular and molecular basis of this phenotype was delineated by showing that low levels of human ECSIT protein led to a marked reduction in assembly and activity of mitochondrial complex I with impaired oxidative phosphorylation and reduced production of ATP. Cardiac tissue from humanized hECSIT mice also showed reduced mitochondrial fusion and more fission but impaired clearance of fragmented mitochondria. A cardiomyocyte-intrinsic role for Ecsit in mitochondrial function and cardioprotection is also demonstrated. We also show that cardiac fibrosis and damage in humans correlated with low expression of human ECSIT. In summary, our findings identify a role for ECSIT in cardioprotection, while generating a valuable experimental model to study mitochondrial dysfunction and cardiac pathophysiology.

The present study aimed at evaluating the mechanism by which functionality of hepatic stellate cells (HSCs) is modulated by bone marrow stromal cells (BMSCs). Induction of apoptosis in HSCs was found to be caused by directly co-culturing HSCs with BMSCs, where the expression of α-smooth muscle actin (α-SMA) increased significantly in HSCs, along with an increase in their proliferation rate. Additionally, expression of Hes1 and Notch1 in HSCs co-cultured with BMSCs increased significantly at both protein and mRNA levels. Blocking of the notch signaling pathway (NSP) either by Notch1 siRNA or by DAPT treatment increased the proliferation rate while decreasing apoptosis and led to activation of the NF-κB signaling pathway in HSCs co-cultured with BMSCs. These effects were found to be reversed in HSCs overexpressing IκB S32/S36 mutants. The Notch signaling-mediated cell-cell contact was partially involved in the significant inhibition of proliferation of HSCs by BMSCs. Additionally, the NF-κB pathway was found to be responsible for NSP-mediated inhibition of growth of HSCs in the co-culture system. Thus, BMSCs might have a potential therapeutic significance in treating hepatic fibrosis.

Hsp70 is an evolutionarily conserved chaperone involved in maintaining protein homeostasis during normal growth and upon exposure to stresses. Mutations in the β6/β7 region of the substrate-binding domain (SBD) disrupt the SBD hydrophobic core resulting in impairment of the heat-shock response and prion propagation in yeast. To elucidate the mechanisms behind Hsp70 loss of function due to disruption of the SBD, we undertook targeted mutational analysis of key residues in the β6/β7 region. We demonstrate the critical functional role of the F475 residue across yeast cytosolic Hsp70-Ssa family. We identify the size of the hydrophobic side chain at 475 as the key factor in maintaining SBD stability and functionality. The introduction of amino acid variants to either residue 475, or close neighbor 483, caused instability and cleavage of the Hsp70 SBD and subsequent degradation. Interestingly, we found that Hsp70-Ssa cleavage may occur through a vacuolar carboxypeptidase (Pep4)-dependent mechanism rather than proteasomal. Mutations at 475 and 483 result in compromised ATPase function, which reduces protein re-folding activity and contributes to depletion of cytosolic Hsp70 in vivo. The combination of reduced functionality and stability of Hsp70-Ssa results in yeast cells that are compromised in their stress response and cannot propagate the [PSI+ ] prion.

Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production.

Hsp70 is a highly conserved molecular chaperone critical for the folding of new and denatured proteins. While traditional models state that cells respond to stress by upregulating inducible HSPs, this response is relatively slow and is limited by transcriptional and translational machinery. Recent studies have identified a number of post-translational modifications (PTMs) on Hsp70 that act to fine-tune its function. We utilized mass spectrometry to determine whether yeast Hsp70 (Ssa1) is differentially modified upon heat shock. We uncovered four lysine residues on Ssa1, K86, K185, K354 and K562 that are deacetylated in response to heat shock. Mutation of these sites cause a substantial remodeling of the Hsp70 interaction network of co-chaperone partners and client proteins while preserving essential chaperone function. Acetylation/deacetylation at these residues alter expression of other heat-shock induced chaperones as well as directly influencing Hsf1 activity. Taken together our data suggest that cells may have the ability to respond to heat stress quickly though Hsp70 deacetylation, followed by a slower, more traditional transcriptional response.

The allosteric coupling of the highly conserved nucleotide- and substrate-binding domains of Hsp70 has been studied intensively. In contrast, the role of the disordered, highly variable C-terminal region of Hsp70 remains unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD motif binds to the tetratricopeptide-repeat domains of Hsp70 co-chaperones. Here, we discovered that the TVEEVD sequence of Saccharomyces cerevisiae cytoplasmic Hsp70 (Ssa1) functions as a SUMO-interacting motif. A second C-terminal motif of ∼15 amino acids between the α-helical lid and the extreme C terminus, previously identified in bacterial and eukaryotic organellar Hsp70s, is known to enhance chaperone function by transiently interacting with folding clients. Using structural analysis, interaction studies, fibril formation assays, and in vivo functional assays, we investigated the individual contributions of the α-helical bundle and the C-terminal disordered region of Ssa1 in the inhibition of fibril formation of the prion protein Ure2. Our results revealed that although the α-helical bundle of the Ssa1 substrate-binding domain (SBDα) does not directly bind to Ure2, the SBDα enhances the ability of Hsp70 to inhibit fibril formation. We found that a 20-residue C-terminal motif in Ssa1, containing GGAP and GGAP-like tetrapeptide repeats, can directly bind to Ure2, the Hsp40 co-chaperone Ydj1, and α-synuclein, but not to the SUMO-like protein SMT3 or BSA. Deletion or substitution of the Ssa1 GGAP motif impaired yeast cell tolerance to temperature and cell-wall damage stress. This study highlights that the C-terminal GGAP motif of Hsp70 is important for substrate recognition and mediation of the heat shock response.

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI +] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes. Electronic supplementary material The online version of this article (10.1007/s00018-017-2698-3) contains supplementary material, which is available to authorized users.

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI +] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes.

To observe the effect of gene expression and tumorigenicity in hybrid cells of human embryonic stem cells (hESCs) and ovarian cancer cells in vitro and in vivo using a mouse model, and to determine its feasibility in reprogramming tumour cells growth and apoptosis, for a potential exploration of the role of hESCs and tumour cells fusion in the management of ovarian cancer. Stable transgenic hESCs (H1) and ovarian cancer cell line OVCAR-3 were established before fusion, and cell fusion system was established to analyse the related indicators. PTEN expression in HO-H1 cells was higher than those in the parental stem cells and lower than those in parental tumour cells; the growth of OV-H1 (RFP+GFP) hybrid cells with double fluorescence expressions were obviously slower than that of human embryonic stem cells and OVCAR-3 ovarian cancer cells. The apoptosis signal of the OV-H1 hybrid cells was significantly higher than that of the hESCs and OVCAR-3 ovarian cancer cells. In vivo results showed that compared with 7 days, 28 days and 35 days after inoculation of OV-H1 hybrid cells; also, apoptotic cell detection indicated that much stronger apoptotic signal was found in OV-H1 hybrid cells inoculated mouse. The hESCs can inhibit the growth of OVCAR-3 cells in vitro by suppressing p53 and PTEN expression to suppress the growth of tumour that may be achieved by inducing apoptosis of OVCAR-3 cells. The change of epigenetics after fusion of ovarian cancer cells and hESCs may become a novel direction for treatment of ovarian cancer.

Fibroblast growth factor 2 (FGF-2) and its main receptor FGFR1 have been shown to promote hepatic stellate cell (HSC) activation and proliferation. However, scant information is available on the anti-fibrogenic activity of FGFR1 inhibitors. The aim of this study was to assess the impact of a selective FGFR1 tyrosine kinase inhibitor NP603 on HSC proliferation and hepatic fibrosis. We demonstrated that rat primary HSCs secreted significant amounts of FGF-2, and its tyrosine phosphorylation of FGFR1 was attenuated by NP603. NP603 inhibited HSC activaton by measuring the expression of α-smooth muscle actin (α-SMA) and the production of type I collagen using ELISA. Furthermore, NP603 (25 μM) in vitro strongly suppressed HSC growth induced by FGF-2 (10 ng/ml) and FCS. This effect correlated with the suppression of extracellular-regulated kinase (ERK) activity and its downstream targets cyclin D1 and p21. In addition, PO NP603 (20 mg·kg−1·day−1) administration significantly decreased hepatic collagen deposition and α-SMA expression in CCl4-treated rats. Collectively, these studies suggest that selective blocking of the FGFR1-mediated pathway could be a promising therapeutic approach for the treatment of hepatic fibrosis.

Aims Bone marrow-derived mesenchymal stem cells (BMSCs) have been reported in many studies to reduce liver fibrosis. Apart from the paracrine mechanism by which the antifibrotic effects of BMSCs inhibit activated hepatic stellate cells (HSCs), the effects of direct interplay and juxtacrine signaling between the two cell types are poorly understood. The purpose of this study was to explore the underlying mechanisms by which BMSCs modulate the function of activated HSCs. Main methods We show here that BMSCs directly cocultured with HSCs significantly suppressed the proliferation and α-smooth muscle actin (α-SMA) expression of HSCs. Moreover, the Notch1 and Hes1 mRNA levels and the Hes1 protein level in cocultured HSCs were evidently higher than in other models. Blocking the Notch signaling pathway with Notch1 siRNA caused the increased expression of phospho-Akt and greater cell growth of cocultured HSCs. This effect was attenuated by the PI3K inhibitor LY294002. Key findings In conclusion, our results demonstrated that BMSCs remarkably inhibited the proliferation of HSCs through a cell–cell contact mode that was partially mediated by Notch pathway activation. In addition, the PI3K/Akt pathway is involved in HSC growth inhibition by the Notch pathway. Significance These findings demonstrated that BMSCs directly modulate HSCs in vitro via Notch signaling cascades. Our results may provide new insights into the treatment of hepatic fibrosis with BMSCs.