Analysis of cholesterol-recognition motifs of the plasma membrane Ca2+-ATPase.

The plasma membrane Ca2+-ATPase (PMCA) is crucial for the fine tuning of intracellular calcium levels in eukaryotic cells. In this study, we show the presence of CARC sequences in all human and rat PMCA isoforms and we performed further analysis by molecular dynamics simulations. This analysis focuses on PMCA1, containing three CARC motifs, and PMCA4, with four CARC domains. In PMCA1, two CARC motifs reside within transmembrane domains, while the third is situated at the intracellular interface. The simulations depict more stable RMSD values and lower RMSF fluctuations in the presence of cholesterol, emphasizing its potential stabilizing effect. In PMCA4, a distinct dynamic was found. Notably, the total energy differences between simulations with cholesterol and phospholipids are pronounced in PMCA4 compared to PMCA1. RMSD values for PMCA4 indicate a more energetically favorable conformation in the presence of cholesterol, suggesting a robust interaction between CARCs and this lipid in the membranes. Furthermore, RMSF analysis for CARCs in both PMCA isoforms exhibit lower values in the presence of cholesterol compared to POPC alone. The analysis of H-bond occupancy and total energy values strongly suggests the potential interaction of CARCs with cholesterol. Given the crucial role of PMCAs in physiological calcium regulation and their involvement in diverse pathological processes, this study underscores the significance of CARC motifs and their interaction with cholesterol in elucidating PMCA function. These insights into the energetic preferences associated with CARC-cholesterol interactions offer valuable implications for understanding PMCA function in maintaining calcium homeostasis and addressing potential associated pathologies.

CETP-derived Peptide Seq-1, the Key Component of HB-ATV-8 Vaccine Prevents Stress Responses, and Promotes Downregulation of Pro-Fibrotic Genes in Hepatocytes and Stellate Cells.

BACKGROUND: The nasal vaccine HB-ATV-8 has emerged as a promising approach for NAFLD (non-alcoholic fatty liver disease) and atherosclerosis prevention. HB-ATV-8 contains peptide seq-1 derived from the carboxy-end of the Cholesteryl Ester Transfer Protein (CETP), shown to reduce liver fibrosis, inflammation, and atherosclerotic plaque formation in animal models. Beyond the fact that this vaccine induces B-cell lymphocytes to code for antibodies against the seq-1 sequence, inhibiting CETP's cholesterol transfer activity, we have hypothesized that beyond the modulation of CETP activity carried out by neutralizing antibodies, the observed molecular effects may also correspond to the direct action of peptide seq-1 on diverse cellular systems and molecular features involved in the development of liver fibrosis. METHODS: The HepG2 hepatoma-derived cell line was employed to establish an in vitro steatosis model. To obtain a conditioned cell medium to be used with hepatic stellate cell (HSC) cultures, HepG2 cells were exposed to fatty acids or fatty acids plus peptide seq-1, and the culture medium was collected. Gene regulation of COL1A1, ACTA2, TGF-ß, and the expression of proteins COL1A1, MMP-2, and TIMP-2 were studied. AIM: To establish an in vitro steatosis model employing HepG2 cells that mimics molecular processes observed in vivo during the onset of liver fibrosis. To evaluate the effect of peptide Seq-1 on lipid accumulation and pro-fibrotic responses. To study the effect of Seq-1-treated steatotic HepG2 cell supernatants on lipid accumulation, oxidative stress, and pro-fibrotic responses in HSC. RESULTS AND CONCLUSION: Peptide seq-1-treated HepG2 cells show a downregulation of COLIA1, ACTA2, and TGF-ß genes, and a decreased expression of proteins such as COL1A1, MMP-2, and TIMP-2, associated with the remodeling of extracellular matrix components. The same results are observed when HSCs are incubated with peptide Seq-1-treated steatotic HepG2 cell supernatants. The present study consolidates the nasal vaccine HB-ATV-8 as a new prospect in the treatment of NASH directly associated with the development of cardiovascular disease.

Peptide Helix-Y12 as Potential Effector for Peroxisome Proliferator-Activated Receptors.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of lipids and glucose metabolism, and immune response. Therefore, they have been considered pharmacological targets for treating metabolic diseases, such as dyslipidemia, atherosclerosis, and non-alcoholic fatty liver disease. However, the available synthetic ligands of PPARs have mild to significant side effects, generating the necessity to identify new molecules that are selective PPAR ligands with specific biological responses. This study aimed to evaluate some components of the atheroprotective and hepatoprotective HB-ATV-8 nanoparticles [the amphipathic peptide Helix-Y12, thermozeaxanthin, thermozeaxanthin-13, thermozeaxanthin-15, and a set of glycolipids], as possible ligands of PPARs through blind molecular docking. According to the change in free energy upon protein-ligand binding, ∆G b, thermozeaxanthins show a more favorable interaction with PPARs, followed by Helix-Y12. Moreover, Helix-Y12 interacts with most parts of the Y-shaped ligand-binding domain (LBD), surrounding helix 3 of PPARs, and reaching helix 12 of PPARα and PPARγ. As previously reported for other ligands, Tyr314 and Tyr464 of PPARα interact with Helix-Y12 through hydrogen bonds. Several PPARα's amino acids are involved in the ligand binding by hydrophobic interactions. Furthermore, we identified additional PPARs' amino acids interacting with Helix-Y12 through hydrogen bonds still not reported for known ligands. Our results show that, from the studied ligand set, the Helix-Y12 peptide and Tzeaxs have the most significant probability of binding to the PPARs' LBD, suggesting novel ligands for PPARs.

The cholesteryl-ester transfer protein isoform (CETPI) and derived peptides: new targets in the study of Gram-negative sepsis.

BACKGROUND: Sepsis is a syndrome where the dysregulated host response to infection threatens the life of the patient. The isoform of the cholesteryl-ester transfer protein (CETPI) is synthesized in the small intestine, and it is present in human plasma. CETPI and peptides derived from its C-terminal sequence present the ability to bind and deactivate bacterial lipopolysaccharides (LPS). The present study establishes the relationship between the plasma levels of CETPI and disease severity of sepsis due to Gram-negative bacteria. METHODS: Plasma samples from healthy subjects and patients with positive blood culture for Gram-negative bacteria were collected at the Intensive Care Unit (ICU) of INCMNSZ (Mexico City). 47 healthy subjects, 50 patients with infection, and 55 patients with sepsis and septic shock, were enrolled in this study. CETPI plasma levels were measured by an enzyme-linked immunosorbent assay and its expression confirmed by Western Blot analysis. Plasma cytokines (IL-1ß, TNFα, IL-6, IL-8, IL-12p70, IFNγ, and IL-10) were measured in both, healthy subjects, and patients, and directly correlated with their CETPI plasma levels and severity of clinical parameters. Sequential Organ Failure Assessment (SOFA) scores were evaluated at ICU admission and within 24 h of admission. Plasma LPS and CETPI levels were also measured and studied in patients  with liver dysfunction. RESULTS: The level of CETPI in plasma was found to be higher in patients with positive blood culture for Gram-negative bacteria that in control subjects, showing a direct correlation with their SOFA values. Accordingly, septic shock patients showing a high CETPI plasma concentration, presented a negative correlation with cytokines IL-8, IL-1ß, and IL-10. Also, in patients  with liver dysfunction, since higher CETPI levels correlated with a high plasma LPS concentration, LPS neutralization carried out by CETPI might be considered a physiological response that will have to be studied in detail. CONCLUSIONS: Elevated levels of plasma CETPI were associated with disease severity and organ failure in patients  with Gram-negative bacteraemia, defining CETPI as a protein implicated in the systemic response to LPS.

The Influence of Interdisciplinary Work towards Advancing Knowledge on Human Liver Physiology.

The knowledge accumulated throughout the years about liver regeneration has allowed a better understanding of normal liver physiology, by reconstructing the sequence of steps that this organ follows when it must rebuild itself after being injured. The scientific community has used several interdisciplinary approaches searching to improve liver regeneration and, therefore, human health. Here, we provide a brief history of the milestones that have advanced liver surgery, and review some of the new insights offered by the interdisciplinary work using animals, in vitro models, tissue engineering, or mathematical models to help advance the knowledge on liver regeneration. We also present several of the main approaches currently available aiming at providing liver support and overcoming organ shortage and we conclude with some of the challenges found in clinical practice and the ethical issues that have concomitantly emerged with the use of those approaches.

Quantitative Expression of Key Cancer Markers in the AS-30D Hepatocarcinoma Model.

Hepatocellular carcinoma is one of the cancers with the highest mortality rate worldwide. HCC is often diagnosed when the disease is already in an advanced stage, making the discovery and implementation of biomarkers for the disease a critical aim in cancer research. In this study, we aim to quantify the transcript levels of key signaling molecules relevant to different pathways known to participate in tumorigenesis, with special emphasis on those related to cancer hallmarks and epithelial-mesenchymal transition, using as a model the murine transplantable hepatocarcinoma AS-30D. Using qPCR to quantify the mRNA levels of genes involved in tumorigenesis, we found elevated levels for Tgfb1 and Spp1, two master regulators of EMT. A mesenchymal signature profile for AS-30D cells is also supported by the overexpression of genes encoding for molecules known to be associated to aggressiveness and metastatic phenotypes such as Foxm1, C-met, and Inppl1. This study supports the use of the AS-30D cells as an efficient and cost-effective model to study gene expression changes in HCC, especially those associated with the EMT process.

Peptides as Therapeutic Molecules to Neutralize Gram-negative Bacterial Lipopolysaccharides in Sepsis and Septic Shock.

During the last years, infections have become a global health emergency, where the appearance of bacteria highly resistant to traditional antibiotics have set off an alarm worldwide. Moreover, the increased incidence and mortality resulting from its aggravated states, sepsis, and septic shock, have been observed with growing concern. In this context, knowing the need for a new concept for treatment, peptides such as antimicrobial peptides (AMP) and host defense peptides (HDP), have started to show interesting properties in the development of new antimicrobial agents and host response modulatory therapies. Nevertheless, since it is a well-known fact that a peptide-based drug development is a long process that consumes a significant number of resources, recent approaches that tend to mitigate these obstacles, have included the implementation of novel in silico strategies for the optimization of naturally occurring AMP and HDP. In this review, we analyze these strategies that seek to improve not only peptide design, but also production, by including the incorporation of computational biology techniques such as molecular dynamics.

Peptide VSAK maintains tissue glucose uptake and attenuates pro-inflammatory responses caused by LPS in an experimental model of the systemic inflammatory response syndrome: a PET study.

The present investigation using Positron Emission Tomography shows how peptide VSAK can reduce the detrimental effects produced by lipopolysaccharides in Dutch dwarf rabbits, used to develop the Systemic Inflammatory Response Syndrome (SIRS). Animals concomitantly treated with lipopolysaccharides (LPS) and peptide VSAK show important protection in the loss of radiolabeled-glucose uptake observed in diverse organs when animals are exclusively treated with LPS. Treatment with peptide VSAK prevented the onset of changes in serum levels of glucose and insulin associated with the establishment of SIRS and the insulin resistance-like syndrome. Treatment with peptide VSAK also allowed an important attenuation in the circulating levels of pro-inflammatory molecules in LPS-treated animals. As a whole, our data suggest that peptide VSAK might be considered as a candidate in the development of new therapeutic possibilities focused on mitigating the harmful effects produced by lipopolysaccharides during the course of SIRS.

New insights into lipopolysaccharide inactivation mechanisms in sepsis.

The complex pathophysiology of sepsis makes it a syndrome with limited therapeutic options and a high mortality rate. Gram-negative bacteria containing lipopolysaccharides (LPS) in their outer membrane correspond to the most common cause of sepsis. Since the gut is considered an important source of LPS, intestinal damage has been considered a cause and a consequence of sepsis. Although important in the maintenance of the intestinal epithelial cell homeostasis, the microbiota has been considered a source of LPS. Recent studies have started to shed light on how sepsis is triggered by dysbiosis, and an increased inflammatory state of the intestinal epithelial cells, expanding the understanding of the gut-liver axis in sepsis. Here, we review the gut-liver interaction in Gram-negative sepsis, exploring the mechanisms of LPS inactivation, including the recently described contribution of an isoform of the cholesteryl-ester transfer protein (CETPI). Although several key questions remain to be answered when the pathophysiology of sepsis is reviewed, new contributions coming to light exploring the way LPS might be inactivated in vivo, suggest that new applications might soon reach the clinical setting.

Hepatic Accumulation of Hypoxanthine: A Link Between Hyperuricemia and Nonalcoholic Fatty Liver Disease.

BACKGROUND: An elevated level of plasma uric acid has been widely recognized as a risk factor for non-alcoholic fatty liver disease (NAFLD), where oxidative stress and inflammation play an important role in the pathophysiology of the disease. Although the complete molecular mechanisms involved remain unknown, while under physiological conditions uric acid presents antioxidant properties, hyperuricemia has been linked to oxidative stress, chronic low-grade inflammation, and insulin resistance, basic signs of NAFLD. AIM OF STUDY: Employing in vivo experimentation, we aim to investigate whether a high-fat diet rich in cholesterol (HFD), modifies the metabolism of purines in close relationship to molecular events associated with the development of NAFLD. In vitro experiments employing HepG2 cells are also carried out to study the phenomenon of oxidative stress. METHODS: Adult male rabbits were fed for 8 weeks an HFD to induce NAFLD. At the beginning of the experiment and every 15 d until the completion of the study, plasma levels of lipids, lipoproteins, and uric acid were measured. Liver tissue was isolated, and histology performed followed by the biochemical determination of hypoxanthine, protein expression of xanthine oxidoreductase (XOR) by western blot analysis, and xanthine oxidase (XO) activity using an enzymatic kinetic assay. Furthermore, we employed in vitro experimentation studying HepG2 cells to measure the effect of hypoxanthine and H2O2 upon the production of radical oxygen species (ROS), XO activity, and cell viability. RESULTS AND CONCLUSION: Hepatic tissue from rabbits fed the HFD diet showed signs of NAFLD associated with an increased ROS concentration and an altered purine metabolism characterized by the increase in hypoxanthine, together with an apparent equilibrium displacement of XOR towards the xanthine dehydrogenase (XDH) isoform of the enzyme. This protein shift visualized by a western blot analysis, associated with an increase in plasma uric acid and hepatocyte hypoxanthine could be understood as a compensatory series of events secondary to the establishment of oxidative stress associated with the chronic establishment of fatty liver disease.