Membrane Fusogenic Activity Research Articles

Serum lipid profile levels and semen quality: new insights and clinical perspectives for male infertility and men's health.

Several clinical scenarios regulate the final ejaculated semen, which is pivotal to reproductive success. Sperm motility and plasma membrane fusogenic activity primarily rely on the peculiar sperm lipid composition, influenced by the patient's metabolism, genetics, nutritional, environmental status, and concomitant clinical entities such as varicocele. This study aimed to determine the relationship between serum lipid profile and testicular function (semen quality and testosterone levels). This retrospective study uses medical charts of 278 infertile men who attended andrological care between 2000 and 2019. Seminal analysis data, lipid profile, and total serum testosterone were collected. A multiple linear regression analysis was performed to evaluate the influence of the lipid parameters on the seminal variables. Statistical analyses were carried out with p ≤ 0.05 considered statistically significant. Seminal creatine kinase activity (p = 0.024) is negatively related to HDL (p = 0.032) and triglycerides (p = 0.037), while total testosterone (p < 0.0001) and seminal volume (p = 0.046) appeared both to be negatively related to triglycerides (p = 0.030 and p = 0.033, respectively). Medical advice commonly advocated to prevent endothelial dysfunction and cardiovascular disease and improve HDL-cholesterol and triglyceride levels in dyslipidemic patients should also be given to infertile men. Physicians should give patients a thorough assessment, including the blood lipid profile, hormonal status, and routine seminal examinations. We propose a more comprehensive men´s health check-up for the infertile male population, not limited to a simple evaluation of basic sperm parameters.

Hydrophilic Hyperbranched Polymer-Coated siRNA/Polyamidoamine Dendron-Bearing Lipid Complexes Preparation for High Colloidal Stability and Efficient RNAi.

RNA interference (RNAi) using siRNA has gained much attention for use in therapies for cancer and genetic disorders. To establish RNAi-based therapeutics, the development of efficient siRNA nanocarriers is desired. Earlier, we developed polyamidoamine dendron-bearing lipids able to form complexes with nucleic acids as gene vectors. Especially, dendron lipids with unsaturated alkyl chains (DL-G1-U2) induced efficient endosomal escape by membrane fusion, leading to efficient transfection in vitro. For this study, dendron lipids having oleyl/linoleyl groups (DL-G1-U3) were designed to increase membrane fusogenic activity further. Indeed, DL-G1-U3/siRNA complexes achieved higher membrane fusogenic activity and knockdown of the target gene more efficiently than conventional DL-G1-U2/siRNA complexes did. A hydrophilic polymer, hyperbranched polyglycidol lauryl ester (HPG-Lau), was modified further on the surface of DL-G1-U3/siRNA complexes to provide colloidal stability. Surface modification of HPG-Lau increased the colloidal stability in a physiological condition more than complexes without HPG-Lau. Importantly, HPG-Lau-coated DL/siRNA complexes showed identical RNAi effects to those of parental DL/siRNA complexes, whereas the RNAi activity of poly(ethylene glycol)-bearing lipid (PEG-PE)-modified DL/siRNA complexes was hindered completely. Introduction of unsaturated bonds into dendron lipids and selection of suitable hydrophilic polymers for nanocarrier modification are important for obtaining efficient siRNA vectors toward in vivo siRNA delivery.

The DI-DII linker of human parainfluenza virus type 3 fusion protein is critical for the virus.

Human parainfluenza virus type 3 (HPIV3) causes the majority of childhood viral pneumonia around the world. Fusing the viral and target cell membranes is crucial for its entry into target cells, and the fusion process requires the concerted actions of two viral glycoproteins: hemagglutinin-neuraminidase (HN) and fusion (F) protein. After binding to the cell surface receptor, sialic acids, HN triggers F to undergo large conformational rearrangements to execute the fusion process. Although it has been reported that several domains of F had important impacts on regulating the membrane fusion activity, what role the DI-DII linker (residues 369-374, namely L1 linker) of the HPIV3 F protein plays in the fusion process still remains confused. We have obtained three chimeric mutant proteins (Ch-NDV-L1, Ch-MV-L1, Ch-HPIV1-L1) containing the full length of HPIV3 F protein but their corresponding DI-DII linker derived from the F protein of Newcastle disease virus (NDV), Measles virus (MV), and Human parainfluenza virus type 1 (HPIV1), respectively. One deletion mutant protein (De-L1), whose DI-DII linker was deleted, has been established simultaneously. Then vaccinia virus-T7 RNA polymerase transient expression system and standard plasmid system were utilized to express the mutant F proteins in BHK-21 cells. These four mutants were determined for membrane fusogenic activity, cell surface expression level, and total mutant F protein expression. All of them resulted in a significant reduction in fusogenic activity in all steps of cell-cell membrane fusion process. There was no significant difference in cell surface protein expression level for the mutants compared with wild-type F. The mutant proteins with inability in fusogenic activity were all at the form of precursor protein, F0, which were not hydrolyzed by intracellular protease furin. The results above suggest that the involvement of the DI-DII linker region is necessary for the complete fusion of the membranes.

Intestinal surfactant permeation enhancers and their interaction with enterocyte cell membranes in a mucosal explant system

ABSTRACTIntestinal permeation enhancers (PEs) are agents aimed to improve oral delivery of therapeutic drugs with poor bioavailability. The main permeability barrier for oral delivery is the intestinal epithelium, and PEs act to increase the paracellular and/or transcellular passage of drugs. Transcellular passage can be achieved by cell membrane permeabilization and/or by endocytic uptake and subsequent transcytosis. One broad class of PEs is surfactants which act by inserting into the cell membrane, thereby perturbing its integrity, but little is known about how the dynamics of the membrane are affected. In the present work, the interaction of the surfactants lauroyl-L-carnitine, 1-decanoyl-rac-glycerol, and nonaethylene glycol monododecyl ether with the intestinal epithelium was studied in organ cultured pig jejunal mucosal explants. As expected, at 2 mM, these agents rapidly permeabilized the enterocytes for the fluorescent polar tracer lucifer yellow, but surprisingly, they all also blocked both constitutive -and receptor-mediated pathways of endocytosis from the brush border, indicating a complete arrest of apical membrane trafficking. At the ultrastructural level, the PEs caused longitudinal fusion of brush border microvilli. Such a membrane fusogenic activity could also explain the observed formation of vesicle-like structures and large vacuoles along the lateral cell membranes of the enterocytes induced by the PEs. We conclude that the surfactant action of the PEs selected in this study not only permeabilized the enterocytes, but profoundly changed the dynamic properties of their constituent cell membranes.

YqiC of Salmonella enterica serovar Typhimurium is a membrane fusogenic protein required for mice colonization

BackgroundSalmonella enterica serovar Typhimurium is an intracellular bacterial pathogen which can colonize a variety of hosts, including human, causing syndromes that vary from gastroenteritis and diarrhea to systemic disease.ResultsIn this work we present structural information as well as insights into the in vivo function of YqiC, a 99-residue protein of S. Typhimurium, which belongs to the cluster of the orthologous group 2960 (COG2960). We found that YqiC shares biophysical and biochemical properties with Brucella abortus BMFP, the only previously characterized member of this group, such as a high alpha helix content, a coiled-coil domain involved in trimerization and a membrane fusogenic activity in vitro. In addition, we demonstrated that YqiC localizes at cytoplasmic and membrane subcellular fractions, that a S. Typhimurium yqiC deficient strain had a severe attenuation in virulence in the murine model when inoculated both orally and intraperitoneally, and was impaired to replicate at physiological and high temperatures in vitro, although it was still able to invade and replicate inside epithelial and macrophages cell lines.ConclusionThis work firstly demonstrates the importance of a COG2960 member for pathogen-host interaction, and suggests a common function conserved among members of this group.

Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase Cι to Associate with Microtubules and to Recruit Dynein

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Ciota (aPKCiota) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCiota in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCiota were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCiota. Because GAPDH binds to the carboxyl terminus of alpha-tubulin, we characterized the distribution of tyrosinated/detyrosinated alpha-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated alpha-tubulin; however, aPKCiota was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCiota-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.

Brucella abortus MFP: A Trimeric Coiled-Coil Protein with Membrane Fusogenic Activity

The bacterial genus Brucella consists of a group of facultative intracellular pathogens which produces abortion and infertility in animals and a chronic debilitating febrile illness in humans. BMFP is a basic protein of Brucella abortus that belongs to a highly conserved group of homologue proteins of unknown structure and function in proteobacteria (COG2960). In this study, we report the structural and biochemical characterization of this protein. We found that BMFP has two structural domains: a carboxyl-terminal coiled-coil domain through which the protein self-associates as a trimer and a natively disordered amino-terminal domain which has propensity to adopt an amphipathic alpha-helical structure. This natively unfolded domain undergoes a structural rearrangement from unfolded to alpha-helix in the presence of high ionic strength, acidic pH, detergents, and phospholipid vesicles. Moreover, we demonstrated that the interaction of BMFP with phospholipid vesicles promotes in vitro membrane fusion. These results contribute to the elucidation of the structural and functional properties of this protein and its homologues present in most proteobacteria.

Membrane Fusogenic Activity of the Alzheimer's Peptide Aβ(1–42) Demonstrated by Small-Angle Neutron Scattering

Amyloid-β peptide (Aβ) is considered a triggering agent of Alzheimer's disease. In relation to a therapeutic treatment of the disease, the interaction of Aβ with the cell membrane has to be elucidated at the molecular level to understand its mechanism of action. In previous works, we had ascertained by neutron diffraction on stacked lipid multilayers that a toxic fragment of Aβ is able to penetrate and perturb the lipid bilayer. Here, the influence of Aβ(1–42), the most abundant Aβ form in senile plaques, on unilamellar lipid vesicles of phospholipids is investigated by small-angle neutron scattering. We have used the recently proposed separated form factor method to fit the data and to obtain information about the vesicle diameter and structure of the lipid bilayer and its change upon peptide administration. The lipid membrane parameters were obtained with different models of the bilayer profile. As a result, we obtained an increase in the vesicle radii, indicating vesicle fusion. This effect was particularly enhanced at pH 7.0 and at a high peptide/lipid ratio. At the same time, a thinning of the lipid bilayer occurred. A fusogenic activity of the peptide may have very important consequences and may contribute to cytotoxicity by destabilizing the cell membrane. The perturbation of the bilayer structure suggests a strong interaction and/or insertion of the peptide into the membrane, although its localization remains beyond the limit of the experimental resolution.

The SARS-CoV S glycoprotein: expression and functional characterization

We have cloned, expressed, and characterized the full-length and various soluble fragments of the SARS-CoV (Tor2 isolate) S glycoprotein. Cells expressing S fused with receptor-expressing cells at neutral pH suggesting that the recombinant glycoprotein is functional, its membrane fusogenic activity does not require other viral proteins, and that low pH is not required for triggering membrane fusion; fusion was not observed at low receptor concentrations. S and its soluble ectodomain, S e, were not cleaved to any significant degree. They ran at about 180–200 kDa in SDS gels suggesting post-translational modifications as predicted by previous computer analysis and observed for other coronaviruses. Fragments containing the N-terminal amino acid residues 17–537 and 272–537 but not 17–276 bound specifically to Vero E6 cells and purified soluble receptor, ACE2, recently identified by M. Farzan and co-workers [Nature 426 (2003) 450–454]. Together with data for inhibition of binding by antibodies developed against peptides from S, these findings suggest that the receptor-binding domain is located between amino acid residues 303 and 537. These results also confirm that ACE2 is a functional receptor for the SARS virus and may help in the elucidation of the mechanisms of SARS-CoV entry and in the development of vaccine immunogens and entry inhibitors.