The Helicobacter pylori HspR-Modulator CbpA Is a Multifunctional Heat-Shock Protein

The medically important human pathogen Helicobacter pylori relies on a collection of highly conserved heat-shock and chaperone proteins to preserve the integrity of cellular polypeptides and to control their homeostasis in response to external stress and changing environmental conditions. Among this set of chaperones, the CbpA protein has been shown to play a regulatory role in heat-shock gene regulation by directly interacting with the master stress-responsive repressor HspR. Apart from this regulatory role, little is known so far about CbpA functional activities. Using biochemistry and molecular biology approaches, we have started the in vitro functional characterization of H. pylori CbpA. Specifically, we show that CbpA is a multifunctional protein, being able to bind DNA and to stimulate the ATPase activity of the major chaperone DnaK. In addition, we report a preliminary observation suggesting that CbpA DNA-binding activity can be affected by the direct interaction with the heat-shock master repressor HspR, supporting the hypothesis of a reciprocal crosstalk between these two proteins. Thus, our work defines novel functions for H. pylori CbpA and stimulates further studies aimed at the comprehension of the complex regulatory interplay among chaperones and heat-shock transcriptional regulators

The heat-shock response of Helicobacter pylori: genomic and molecular characterization of the master repressor HspR

The heat-shock response (HSR) induces the expression of heat-shock proteins, ensuring the bacterial cells to adapt to hostile environmental conditions during stress. In Helicobacter pylori, the regulation of the principal genes encoding the heat-shock proteins is under the transcriptional control of two repressor proteins named HspR and HrcA, with the former acting as the master regulator of the circuit. In order to further characterize the HspR regulon and deepen our understanding of HSR in H. pylori we used global transcriptome analysis in combination with Chromatin ImmunoPrecipitation of in vivo HspR genomic binding sites. These data showed that HspR is involved in the regulation of different cellular crucial functions directly controlling a limited set of target genes. Moreover, to provide further details on HspR-DNA interactions on its genomic targets we performed hydroxyl-radical footprinting experiments. This analysis revealed a peculiar periodic pattern of DNA protection. From a nucleotide sequence alignment of HspR binding sites, DNA sequences with similarities to the HAIR motif were identified. Through sitedirected mutagenesis we demonstrated in vitro that the HAIR-like motif is essential for the HspR binding to its own promoter region and that non-conserved nucleotides flanking the HAIR-like motif are necessary for the HspR complete binding on its operator sequence. An important role in resistance against environmental stresses is also played by the ATP-dependent caseinolytic proteases (Clp), a class of serine proteases involved in protein quality control as well as in degradation of regulatory proteins. In order to get more information about the role played by the Clp proteases in H. pylori and to directly identify their protein substrates, we implemented a strategy to express in vivo a proteolytic inactive form of ClpP, the catalytic subunit of this class of proteases, that will retain but not degrade substrates translocated into its proteolytic chamber

Microbial characterization of sourdough for sweet baked products in the Campania region (southern Italy) by a polyphasic approach

The microflora of nine sourdoughs used for sweet bakery products underwent preliminary microbiological characterization using lactic acid bacteria (LAB) and yeast enumeration. Five sourdough samples were submitted for microbial identification by culture-dependent techniques employing 16S and 26S rRNA genes sequencing, as well as a culture-independent technique using PCR-DGGE analysis. The LAB species isolated belonged principally to facultative heterofermentative Lactobacillus spp., Leuconostoc spp., and Lactococcus spp. Yeast strains were identified as Saccharomyces cerevisiae, with one exception represented by a strain belonging to Metschnikowia pulcherrima. PCR-DGGE analysis allowed the identification of Streptococcus thermophilus, Lactobacillus sakei, Weissella groceries and Lactobacillus sanfranciscensis among lactic acid bacteria and Saccharomyces cerevisiae and Metschnikowia pulcherrima among yeasts. This polyphasic approach highlighted different levels of biodiversity, from two to eight different typical LAB species, always associated to Saccharomyces cerevisiae, that could be selected to be specifically used in naturally fermented brioche and cornetto preparation

Targeting unfolded protein response reverts ER stress and ER Ca2+ homeostasis in cardiomyocytes expressing the pathogenic variant of Lamin A/C R321X

Background: We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. Methods: HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. Results: We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. Conclusions: We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes

Differential expression of sphingolipid metabolizing enzymes in spontaneously hypertensive rats: a possible substrate for susceptibility to brain and kidney damage

Alterations in the metabolism of sphingolipids, a class of biologically active molecules in cell membranes with direct effect on vascular homeostasis, are increasingly recognized as important determinant in different vascular disorders. However, it is not clear whether sphingolipids are implicated in the pathogenesis of hypertension-related cerebrovascular and renal damage. In this study, we evaluated the existence of possible abnormalities related to the sphingolipid metabolism in the brain and kidneys of two well validated spontaneously hypertensive rat strains, the stroke-prone (SHRSP) and the stroke-resistant (SHRSR) models, as compared to the normotensive Wistar Kyoto (WKY) rat strain. Our results showed a global alteration in the metabolism of sphingolipids in both cerebral and renal tissues of both hypertensive strains as compared to the normotensive rat. However, few defects, such as reduced expression of enzymes involved in the metabolism/catabolism of sphingosine-1-phosphate and in the de novo biosynthetic pathways, were exclusively detected in the SHRSP. Although further studies are necessary to fully understand the significance of these findings, they suggest that defects in specific lipid molecules and/or their related metabolic pathways may likely contribute to the pathogenesis of hypertensive target organ damage and may eventually serve as future therapeutic targets to reduce the vascular consequences of hypertension

A Novel Vasoactive Peptide “PG1” from Buffalo Ice-Cream Protects from Angiotensin-Evoked High Blood Pressure

Arterial hypertension is the most important risk factor for cardiovascular diseases, myocardial infarction, heart failure, renal failure and peripheral vascular disease. In the last decade, milk-derived bioactive peptides have attracted attention for their beneficial cardiovascular properties. Methods: Here, we combined in vitro chemical assay such as LC-MS/MS analysis of buffalo ice cream, ex vivo vascular studies evaluating endothelial and smooth muscle responses using pressure myograph, and translational assay testing in vivo the vascular actions of PG1 administration in murine models. Results: We demonstrate that a novel buffalo ice-cream-derived pentapeptide “QKEPM”, namely PG1, is a stable peptide that can be obtained at higher concentration after gastro-intestinal digestions (GID) of buffalo ice-cream (BIC). It owns potent vascular effect in counteract the effects of angiotensin II-evoked vasoconstriction and high blood pressure levels. Its effects are mediated by the inhibitory effect on AT1 receptor leading to a downregulation of p-ERK½/Rac1-GTP and consequent reduction of oxidative stress. Conclusions: These results strongly candidate PG1, as a novel bioactive peptide for the prevention and management of hypertension, thus expanding the armamentarium of preventive strategies aimed at reducing the incidence and progression of hypertension and its related cardiovascular complication

Traceability and authentication in agri-food production: a multivariate approach to the characterization of the Italian food excellence elephant garlic (Allium ampeloprasum L.), a vasoactive nutraceutical

A research platform for food authentication was set up by combining stable isotope ratio analysis, metabolomics by gas and liquid mass-spectrometry and NMR investigations, chemometric analyses for food excellences. This multi-analytical approach was tested on samples of elephant garlic (Allium ampeloprasum L.), a species belonging to the same genus of common garlic (Allium ampeloprasum L.), mainly produced in southern Tuscany-(Allium ampeloprasum). The isotopic composition allowed the product to be geographically characterized. Flavonoids, like (+)-catechin, cinnamic acids, quercetin glycosides were identified. The samples showed also a significant amount of dipeptides, sulphur-containing metabolites and glutathione, the latter of which could be considered a molecular marker of the analyzed elephant garlic. For nutraceutical profiling to reach quality labels, extracts were investigated in specific biological assays, displaying interesting vasorelaxant properties in rat aorta by mediating nitric oxide release from the endothelium and exhibited positive inotropic and negative chronotropic effects in rat perfused hear

Peptidomimetics as potent dual SARS-CoV-2 cathepsin-L and main protease inhibitors: In silico design, synthesis and pharmacological characterization

In this paper we present the design, synthesis, and biological evaluation of a new series of peptidomimetics acting as potent anti-SARS-CoV-2 agents. Starting from our previously described Main Protease (MPro) and Papain Like Protease (PLPro) dual inhibitor, CV11, here we disclose its high inhibitory activity against cathepsin L (CTSL) (IC50 = 19.80 ± 4.44 nM), an emerging target in SARS-CoV-2 infection machinery. An in silico design, inspired by the structure of CV11, led to the development of a library of peptidomimetics showing interesting activities against CTSL and Mpro, allowing us to trace the chemical requirements for the binding to both enzymes. The screening in Vero cells infected with 5 different SARS-CoV-2 variants of concerns, highlighted sub-micromolar activities for most of the synthesized compounds (13, 15, 16, 17 and 31) in agreement with the enzymatic inhibition assays results. The compounds showed lack of activity against several different RNA viruses except for the 229E and OC43 human coronavirus strains, also characterized by a cathepsin-L dependent release into the host cells. The most promising derivatives were also evaluated for their chemical and metabolic in-vitro stability, with derivatives 15 and 17 showing a suitable profile for further preclinical characterization