Ivan Haralampiev

Cholesterol’s Aliphatic Side Chain Modulates Membrane Properties†

The influence of cholesterols alkyl side chain on membrane properties was studied using a series of synthetic cholesterol derivatives without a side chain or with a branched side chain consisting of 5 to 14 carbon atoms. Cholesterols side chain is crucial for all membrane properties investigated and therefore essential for the membrane properties of eukaryotic cells.

Membrane bound α-synuclein is fully embedded in the lipid bilayer while segments with higher flexibility remain.

Cellular pathways involving α‐synuclein (αS) seem to be causative for development of Parkinsons disease. Interactions between αS and lipid membranes appear to be important for the physiological function of the protein and influence the pathological aggregation of αS leading to the formation of amyloid plaques. Upon membrane binding the unstructured αS folds into amphipathic helices. In our work we characterized the penetration depth and probed the local environment of Trp‐residues introduced along the αS sequence. We could show that while the entire helix is well embedded in the lipid bilayer, segments with a shallower penetration and supposable higher flexibility exist.

Membrane properties of cholesterol analogs with an unbranched aliphatic side chain.

The interactions between cholesterol and other membrane molecules determine important membrane properties. It was shown that even small changes in the molecular structure of cholesterol have a crucial influence on these interactions. We recently reported that in addition to alterations in the tetracyclic ring structure, the iso-branched side chain of cholesterol also has a significant impact on membrane properties (Scheidt et al., 2013). Here we used synthetic cholesterol analogs to investigate the influence of an unbranched aliphatic side chain of different length. The (2)H NMR order parameter of the phospholipid chains and therefore the molecular packing of the phospholipid molecules shows a significant dependence on the sterols alkyl side chain length, while, membrane permeation studied by a dithionite ion permeation assay and lateral diffusion measured by (1)H MAS pulsed field gradient NMR are less influenced. To achieve the same molecular packing effect similar to that of an iso-branched aliphatic side chain, a longer unbranched side chain (n-dodecyl instead of n-octyl) at C17 of cholesterol is required. Obviously, sterols having a branched iso-alkyl chain with two terminal methyl groups exhibit altered cholesterol-phospholipid interactions compared to analogous molecules with a straight unbranched chain.

Amyloid-β(1-42) aggregation initiates its cellular uptake and cytotoxicity

The accumulation of amyloid β peptide(1–42) (Aβ(1–42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Aβ(1–42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Aβ may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Aβ endocytosis. We visualized aggregate formation of fluorescently labeled Aβ(1–42) and tracked its internalization by human neuroblastoma cells and neurons. β-Sheet-rich Aβ(1–42) aggregates entered the cells at low nanomolar concentration of Aβ(1–42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Aβ(1–42) aggregates to form. By uncoupling membrane binding from internalization, we found that Aβ(1–42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of β-sheet-rich aggregates is a prerequisite for Aβ(1–42) uptake and cytotoxicity.

Recruitment of SH-Containing peptides to lipid and biological membranes through the use of a palmitic acid functionalized with a Maleimide Group

This study presents a novel and easily applicable approach to recruit sulfhydryl-containing biomolecules to membranes by using a palmitic acid which is functionalized with a maleimide group. Notably, this strategy can also be employed with preformed (biological) membranes. The applicability of the assay is demonstrated by characterizing the binding of a Rhodamine-labeled peptide to lipid and cellular membranes using methods of fluorescence spectroscopy, lifetime measurement, and microscopy. Our approach offers new possibilities for preparing biologically active liposomes and manipulating living cells.

Interaction of fluorescent phospholipids with cyclodextrins.

Fluorescent analogs of phospholipids are often employed to investigate the structure and dynamics of lipids in membranes. Some of those studies have used cyclodextrins e.g., to modulate the lipid phase. However, the role of the fluorescence moiety of analogs for the interaction between cyclodextrins and fluorescent lipids has not been investigated so far in detail. Therefore, in the present study the interaction of various fluorescent phospholipid analogs with methylated α-, β- and γ- cyclodextrins was investigated. The analogs differed in their structure, in the length of the fatty acyl chain, in the position of the fluorescence group, and in the attached fluorescence moiety (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or dipyrrometheneboron difluoride (BODIPY)). In aqueous buffer, cyclodextrins bind fluorescent lipids disturbing the organization of the analogs. When incorporated into lipid vesicles, analogs are selectively extracted from the membrane upon addition of cyclodextrins. The results show that the interaction of cyclodextrins with fluorescent phospholipids depends on the cyclodextrin species, the fluorescence moiety and the phospholipid structure. The presented data should be of interest for studies using fluorescent phospholipids and cyclodextrins, since the interaction between the fluorescence group and the cyclodextrin may interfere with the process(es) under study.

Influenza A virus nucleoprotein targets subnuclear structures.

The Influenza A virus nucleoprotein (NP) is the major protein component of the genomic viral ribonucleoprotein (vRNP) complexes, which are the replication‐ and transcription‐competent units of Influenza viruses. Early during infection, NP mediates import of vRNPs into the host cell nucleus where viral replication and transcription take place; also newly synthesized NP molecules are targeted into the nucleus, enabling coreplicational assembly of progeny vRNPs. NP reportedly acts as regulatory factor during infection, and it is known to be involved in numerous interactions with host cell proteins. Yet, the NP‐host cell interplay is still poorly understood.

Membrane properties of hydroxycholesterols related to the brain cholesterol metabolism

Compared to cholesterol, hydroxycholesterols contain an additional hydroxy group in the alkyl chain and are able to efficiently cross the brain–blood barrier. Therefore, they are responsible for the sterol transfer between brain and circulation. The current study compares the membrane properties of several hydroxycholesterols with those of cholesterol using 2H NMR spectroscopy, a membrane permeability assay, and fluorescence microscopy experiments. It is shown that hydroxycholesterols do not exert the unique impact on membrane properties characteristic for cholesterol with regard to the influence on lipid chain order, membrane permeability and formation of lateral domains.

Lipid dynamics in boar sperm studied by advanced fluorescence imaging techniques

The (re)organization of membrane components is of special importance to prepare mammalian sperm to fertilization. Establishing suitable methods to examine physico-chemical membrane parameters is of high interest. We characterized the behavior of fluorescent (NBD) analogs of sphingomyelin (SM), phosphatidylserine (PS), and cholesterol (Ch) in the acrosomal and postacrosomal macrodomain of boar sperm. Due to their specific transverse membrane distribution, a leaflet-specific investigation of membrane properties is possible. The behavior of lipid analogs in boar sperm was investigated by fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS). The results were compared with regard to the different temporal and spatial resolution of the methods. For the first time, fluorescence lifetimes of lipid analogs were determined in sperm cell membrane and found to be in a range characteristic for the liquid-disordered phase in artificial lipid membranes. FLIM analyses further indicate a more fluid microenvironment of NBD-Ch and NBD-PS in the postacrosomal compared to the acrosomal region. The concept of a more fluid cytoplasmic leaflet is supported by lower fluorescence lifetime and higher average D values (FCS) for NBD-PS in both head compartments. Whereas FLIM analyses did not indicate coexisting distinct liquid-ordered and -disordered domains in any of the head regions, comparisons between FRAP and FCS measurements suggest the incorporation of NBD-SM as well as NBD-PS in postacrosomal subpopulations with different diffusion velocity. The analog-specific results indicate that the lipid analogs used are suitable to report on the various physicochemical properties of different microenvironments.

Synthesis and Characterization of a New Bifunctionalized, Fluorescent, and Amphiphilic Molecule for Recruiting SH-Containing Molecules to Membranes

This study describes the synthesis and characterization of an amphiphilic construct intended to recruit SH‐containing molecules to membranes. The construct consists of 1) an aliphatic chain to enable anchoring within membranes, 2) a maleimide moiety to react with the sulfhydryl group of a soluble (bio)molecule, and 3) a fluorescence moiety to allow the construct to be followed by fluorescence spectroscopy and microscopy. It is shown that the construct can be incorporated into preformed membranes, thus allowing application of the approach with biological membranes. The close proximity between the fluorophore and the maleimide moiety within the construct causes fluorescence quenching. This allows monitoring of the reaction with SH‐containing molecules by measurement of increases in fluorescence intensity and lifetime. Notably, the construct distributes into laterally ordered membrane domains of lipid vesicles, which is probably triggered by the length of its membrane anchor. The advantages of the new construct can be employed for several biological, biotechnological, and medicinal applications.