Renato Longhi

Triglycerides Are Major Determinants of Cholesterol Esterification/Transfer and HDL Remodeling in Human Plasma

Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) are responsible for the esterification of cell-derived cholesterol and for the transfer of newly synthesized cholesteryl esters (CE) from HDL to apoB-containing lipoproteins in human plasma. LCAT and CETP are also crucial factors in HDL remodeling, a process by which HDL particles with a high capacity for cell cholesterol uptake are generated in plasma. In the present study, cholesterol esterification and transfer were evaluated in 60 patients with isolated hypercholesterolemia (HC, n = 20) and isolated (HTG, n = 20) or mixed hypertriglyceridemia (MHTG, n = 20) and in 20 normolipidemic healthy individuals (NL). Cholesterol esterification rate (CER) and net CE transfer rate (CETR) were measured in whole plasma. LCAT and CETP concentrations were determined by specific immunoassays. HDL remodeling was analyzed by monitoring changes in HDL particle size distribution during incubation of whole plasma at 37 degrees C. Mean CER and CETR were 48% and 73% higher, respectively, in hypertriglyceridemic (HTG + MHTG) versus normotriglyceridemic individuals. HDL remodeling was also significantly accelerated in plasma from hypertriglyceridemic patients. Strong positive correlations were found in the total sample between plasma and VLDL triglyceride levels and CER (r = .722 and r = .642, respectively), CETR (r = .510 and r = .491, respectively), and HDL remodeling (r = .625 and r = .620, respectively). No differences in plasma LCAT and CETP concentrations were found among the various groups except for a tendency toward higher CETP levels in hypercholesterolemic patients (+51% in MHTG and +20% in HC) versus control subjects (NL). By stepwise regression analysis, VLDL triglyceride level was the sole significant predictor of CER and CETR and contributed significantly together with baseline HDL particle distribution to HDL remodeling. These results indicate that plasma triglyceride level is a major factor in the regulation of cholesterol esterification/transfer and HDL remodeling in human plasma, whereas LCAT/CETP concentrations play a minor role in the modulation of reverse cholesterol transport.

Protection of Sinorhizobium against Host Cysteine-Rich Antimicrobial Peptides Is Critical for Symbiosis

A bacterial membrane protein, BacA, protects Sinorhizobium meliloti against the antimicrobial activity of host peptides, enabling the peptides to induce bacterial persistence rather than bacterial death.

Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis

Significance Sinorhizobium meliloti and its legume hosts establish a symbiosis in which bacterial fixed nitrogen is exchanged for plant carbon compounds. We study this symbiosis because it is agriculturally and ecologically important and to identify mechanisms used in host–microbe interactions. S. meliloti is internalized in specialized host nodule cells that then use small, cysteine-rich peptides to drive their differentiation into polyploid cells that fix nitrogen. We found that a representative host peptide blocks cell division, in part by eliciting significant changes in the expression of genes that regulate the cell cycle and mediate cell division. We also found that the peptide activated pathways conserved in related pathogens. Our study provides insights into how host peptides cause differentiation of S. meliloti during symbiosis. The α-proteobacterium Sinorhizobium meliloti establishes a chronic intracellular infection during the symbiosis with its legume hosts. Within specialized host cells, S. meliloti differentiates into highly polyploid, enlarged nitrogen-fixing bacteroids. This differentiation is driven by host cells through the production of defensin-like peptides called “nodule-specific cysteine-rich” (NCR) peptides. Recent research has shown that synthesized NCR peptides exhibit antimicrobial activity at high concentrations but cause bacterial endoreduplication at sublethal concentrations. We leveraged synchronized S. meliloti populations to determine how treatment with a sublethal NCR peptide affects the cell cycle and physiology of bacteria at the molecular level. We found that at sublethal levels a representative NCR peptide specifically blocks cell division and antagonizes Z-ring function. Gene-expression profiling revealed that the cell division block was produced, in part, through the substantial transcriptional response elicited by sublethal NCR treatment that affected ∼15% of the genome. Expression of critical cell-cycle regulators, including ctrA, and cell division genes, including genes required for Z-ring function, were greatly attenuated in NCR-treated cells. In addition, our experiments identified important symbiosis functions and stress responses that are induced by sublethal levels of NCR peptides and other antimicrobial peptides. Several of these stress-response pathways also are found in related α-proteobacterial pathogens and might be used by S. meliloti to sense host cues during infection. Our data suggest a model in which, in addition to provoking stress responses, NCR peptides target intracellular regulatory pathways to drive S. meliloti endoreduplication and differentiation during symbiosis.

Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells

The γ-aminobutyric acid transporter (GAT-1) isoform of the γ-aminobutyric acid and the betaine (BGT) transporters exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G., and Caplan, M. J. (1994) J. Biol. Chem. 269, 4668-4674). We have investigated the presence of sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric transporters. Whereas truncated GAT-1 (ΔC-GAT) maintained the original functional activity and apical localization, either the removal (ΔC-myc BGT) or the substitution (BGS chimera) of the cytosolic tail of BGT generated proteins that accumulated in the endoplasmic reticulum. Moreover, we have found that the cytosolic tail of BGT redirected apical proteins, the polytopic GAT-1 (GBS chimera) and the monotopic human nerve growth factor receptor, to the basolateral surface. These results suggest the presence of basolateral sorting information in the cytosolic tail of BGT. We have further shown that information necessary for the exit of BGT from the endoplasmic reticulum and for the basolateral localization of the GBS chimera is contained in a short segment, rich in basic residues, within the cytosolic tail of BGT.

Role of Cysteine Residues and Disulfide Bonds in the Activity of a Legume Root Nodule-specific, Cysteine-rich Peptide

Background: Legume antimicrobial peptides (AMPs) mediate Sinorhizobium meliloti bacteroid differentiation. Results: Cysteine replacements and disulfide bond modifications influence the antimicrobial activity of a legume AMP and its ability to mediate S. meliloti bacteroid differentiation. Conclusion: Specific changes to legume AMPs influence their activity against S. meliloti. Significance: Understanding the relationship of AMPs in S. meliloti bacteroid differentiation is fundamental for nitrogen fixation and legume growth. The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S–S) bridges. However, the natural configuration of NCR AMP S–S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S–S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S–S bridges from cysteines 1–2, 3–4 to 1–3, 2–4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.

Discrete regions of HIV-1 gp41 defined by syncytia-inhibiting affinity-purified human antibodies.

ObjectiveFine mapping of HIV-1 gp41 fusion-critical sites. Design and methodsAntibodies from human HIV-1-positive sera were affinity-purified on a panel of synthetic overlapping peptides spanning residues 526–682 of the extracellular portion of HIV-1 gp41. The syncytium-inhibiting capacity of the immunopurified antibodies and their differential reactivity on the synthetic peptides were tested. ResultsThis approach enabled the identification of residues 583–591 (ARILAVERY), 595–599 (QQLLG), 603–609 (CSGKLIC) and 664–673 (ELLELDKWAS) as possibly involved in the fusion process. Reduction in the anti-ARILAVERY, anti-CSGKLIC and anti-ELLELDKWAS antibody titres and frequencies correlates with disease progression. Syncytia-inhibition capacity of sera did not correlate with the presence of high-titre antibodies reacting with any of the peptides tested, suggesting that most fusion-affecting antibodies are not directed towards gp41. ConclusionsThis strategy may be relevant for understanding the contribution of anti-gp41 antibodies in protecting against the pathogenic effects of the virus and in the design of an effective env vaccine.

Protein Kinase C-mediated Phosphorylation of the BGT1 Epithelial γ-Aminobutyric Acid Transporter Regulates Its Association with LIN7 PDZ Proteins A POST-TRANSLATIONAL MECHANISM REGULATING TRANSPORTER SURFACE DENSITY

The Na/Cl-dependent BGT1 transporter has osmoprotective functions by importing the small osmolyte betaine into the cytosol of renal medullary epithelial cells. We have demonstrated previously that the surface localization of the transporter in Madin-Darby canine kidney cells depends on its association with the LIN7 PDZ protein through a PDZ target sequence in the last 5 residues of the transporter (-KETHL). Here we describe a protein kinase C (PKC)-mediated mechanism regulating the association between BGT1 and LIN7. Reduced transport activity paralleled by the intracellular relocalization of the transporter was observed in response to the PKC activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. This activation caused clathrin-dependent internalization of the transporter and its targeting to a recycling compartment that contains the truncated transporter lacking the LIN7 binding motif (BGTΔ5) but not the LIN7 partner of BGT1. The decreased association between BGT1 and LIN7 was demonstrated further by coimmunoprecipitation studies and in vitro binding to recombinant LIN7 fusion protein. The TPA treatment induced phosphorylation of surface BGT1 on serine and threonine residues. However, a greater increase in phosphothreonines than phosphoserines was measured in the wild type transporter, whereas the opposite was true in the BGTSer mutant in which a serine replaced the threonine 612 in the LIN7 association motif (-KESHL). No similar increase in relative phosphoserines or phosphothreonines was found in the BGTΔ5 transporter. Moreover, phosphorylation of threonine 612 in a BGT COOH-terminal peptide impaired its association with recombinant LIN7. Taken together, these data demonstrate that the post-translational regulation of BGT1 surface density is a result of transporter phosphorylation and that threonine 612 is an essential residue in this PKC-mediated regulation.