Biophysical characterization of reactions associated with reverse cholesterol transport

This thesis aimed at improving our understanding of reactions relevant in the reverse cholesterol transport (RCT). RCT facilitates cholesterol homeostasis and is the most important pathway involved in cardiovascular disease. For this purpose three different projects were chosen. Thermodynamics of protein self-association and unfolding was characterized in detail at the example of Apolipoprotein A-1 (Apo A-1). Lipid binding was characterized by means of small peptides that mimic Apo A-1 function. The third project gained insight about the molecular mechanisms of ABCA1`s allocrite flopping. Apo A-1 is the main protein constituent of high density lipoprotein (HDL) and is together with ABCA1 a key player of the RCT. Apolipoprotein A-1 Protein self-association and unfolding are two processes whose understanding is of utmost importance for the development of biological phamaceuticals as oligomerisation may alter functional properties of proteins. Apo A-1 is a perfect candidate for these investigations as it undergoes a concentration dependent self-association process and has high physiological relevance. Even though Apo A-1 is a highly investigated macromolecule, self-association was not investigated in such a comprehensive approach. Additionally, we used highly purified recombinant human Apo A-1, which was generously provided by H.-J. Schönfeld. For analyzing thermodynamics of self-association and thermal unfolding we introduced new theoretical and experimental methods Self-association data was obtained by a combination of high sensitivity micro calorimetry and analytical ultracentrifugation. The dissociation reaction of highly concentrated and thus oligomeric Apo A-1 was followed by injection into buffer in an isothermal titration calorimeter (ITC). Dilution of the sample moved the chemical equilibrium towards monomers. Complementary, this equilibrium was analyzed by data obtained from analytical ultracentrifugation in a sedimentation equilibrium mode. If any, self-association was described in former studies as equilibrium between distinct species, for example between monomers and dimers. We introduced a cooperative self-association model that describes the equilibrium of the protein between each possible oligomer in a concentration dependent manner. Furthermore, we introduced a “binding partition function” that represents the sum of all concentrations found in the system. Together with a dissociation degree of the protein we found a link between thermodynamic data and theory of self-association. The binding partition function describes the statistical properties of the system in thermodynamic equilibrium. Hence, it is independent of the theoretical model that is utilized to describe the reaction. Thermal unfolding of Apo A-1 was followed by circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). We found that melting of Apo A-1 caused a transition of α–helix to β–sheet and random coiled secondary structure and appeared to be highly reversibly up to 75 °C. Thermal unfolding of Apo A-1 and in general of proteins is analyzed almost exclusively with an all-or-none model. As a powerful alternative for higly α–helical proteins such as Apo A 1, we introduced the cooperative Zimm-Bragg theory. Zimm-Bragg theory is commonly used for thermal unfolding of peptides, but fits well to our data and to data of other proteins obtained from literature. Apo A-1 mimetic peptides Apo A-1 was proposed as drug against cardiovascular disease. However, Apo A-1 mimetic peptides are more promising as they have to be administered in much lower dosage and are produced more easily. Understanding their lipid binding properties is essential for the estimation of in vivo effects as well as for formulation and dosage of possible drugs with these peptides. Apo A-1 structure is featured by several amphiphatic class A motif α-helices. Even though it is the main protein component of HDL, thermodynamic characterization of its lipid binding has not been achieved in detail. As a model of Apo A-1 we used two peptides (4F and P), which are featured by class A amphipathic α-helical sequences. 4F showed Apo A-1 mimetic properties in animal models and clinical studies. We used isothermal titration calorimetry to determine thermodynamic parameters of binding to POPC lipid vesicles. In order to understand this reaction several other experimental methods were used. Static and dynamic light scattering illustrated the ability of the peptides to rupture unilamellar vesicles and form micellar-like particles. In contrast, many other peptides such as cell penetrating peptides (CPPs) only partition into the membrane. This finding is in agreement with a 1:1 lipid-to-peptide stochiometry yielded from ITC data analyzed with a model of n identical binding sites. This behavior might have high physiological relevance as possible rupture of cell membranes is unwanted. Circular dichroism experiments yielded insight into structural transitions as part of the driving force of lipid binding. Associated with lipid binding is a transition of the peptide from β–sheet and random coiled to α-helical secondary structure. Tryptophan fluorescence measurements complemented the studies indicating binding to lipids as well. Thermodynamic calculation proved the structural transition of β–sheet and random coiled to α-helix as well as hydrophobic interactions as driving forces of the reaction. Further, we studied binding of the peptide 4F to cholesterol by means of ITC. Our results suggested affinity of 4F towards cholesterol but with lower affinity compared to POPC. This might explain the formation of HDL like particles, mainly consisting of phosphocholine lipids. These particles, in turn, could bind to cholesterol with high affinity. ABCA1 ABCA1 is an ATP binding cassette transporter that flops excess lipids of a cell to the outer membrane leaflet, where it can be picked up by Apo A-1 or HDL particles. Research in the field of ABCA1 is mainly focused on studies in cell culture and in animal models and is therefore rather indirect. Cholesterol efflux by ABCA1 was assumed to be controlled by the copy number of the transporter. The possibility of a direct modulation of the transporter activity by allocrites like in P-glycoprotein (Pgp) as well as the proposed allocrite specificity was rarely investigated in previous studies. Here, we measured the ATPase activity of inside-out vesicles prepared from ABCA1 transfected Human Embryonic Kidney 293 cells by means of a spectroscopic phosphate release assay. Aluminum fluorides were found as strong inhibitor of the nucleotide binding sites (NBD) of ABCA1 in contrast to vanadate. Furthermore, a screening for putative allocrites interacting with the transmembrane domains (TMDs) was performed with numerous compounds. Therewith we found that all compounds with a pegylated chain, a heterocyclic group and a hydrocarbon tail indicated activation of the ABCA1 ATPase

Monomeric versus decameric vanadate in the elucidation of muscle contraction regulation: a kinetic, spectroscopic and structural overview

Vanadium (V) was rediscovered for biology as a “muscle inhibitor factor” when it was found in commercial ATP prepared from equine muscle almost thirty years ago. Since then it has been used as a molecular probe of the mechanisms of several enzyme reactions involving hydrolysis of phosphate ester bonds. Besides acting as a phosphate analogue, vanadate has also the potential to exhibit biological activities through oligomeric vanadate species. Among the vanadate oligomers, decavanadate is one of the most potent inhibitors and has revealed an excellent kinetic and spectroscopic probe. This is particularly relevant for myosin, the major muscle ATPase which along with actin is able to convert the chemical energy of ATP hydrolysis into mechanical work. Apparently, vanadate is able to populate different conformational states of the myosin ATPase cycle depending on its oligomerization state. While monomeric vanadate (VO4 3-) mimics the transition state for the g-phosphate hydrolysis blocking myosin in a pre-power stroke state, decameric vanadate (V10O28 6-) induces the formation of the intermediate myosin·MgATP·V10 complex blocking the actomyosin cycle in a pre-hydrolysis state. These recent findings, that are now reviewed, point out to the importance of taking into account vanadate species variety in studies describing the interaction of vanadate with biological systems and incite the use of decavanadate as a biochemical tool to the elucidation of muscle contraction regulation

Antidiabetic Bis-Maltolato-OxoVanadium(IV): Conversion of inactive trans- to bioactive cis-BMOV for possible binding to target PTP-1B

The postulated transition of Bis-Maltolato-OxoVanadium(IV) (BMOV) from its inactive trans- into its cis-aquo-BMOV isomeric form in solution was simulated by means of computational molecular modeling. The rotational barrier was calculated with DFT – B3LYP under a stepwise optimization protocol with STO-3G, 3-21G, 3-21G*, and 6-31G ab initio basis sets. Our computed results are consistent with reports on the putative molecular mechanism of BMOV triggering the insulin-like cellular response (insulin mimetic) as a potent inhibitor of the protein tyrosine phosphatase-1B (PTP-1B). Initially, trans-BMOV is present in its solid dosage form but in aqueous solution, and during oral administration, it is readily converted into a mixture of “open-type” and “closed-type” complexes of cis-aquo-BMOV under equilibrium conditions. However, in the same measure as the “closed-type” complex binds to the cytosolic PTP-1B, it disappears from solution, and the equilibrium shifts towards the “closed-type” species. In full accordance, the computed binding mode of cis-BMOV is energetically favored over sterically hindered trans-BMOV. In view of our earlier report on prodrug hypothesis of vanadium organic compounds the present results suggest that cis-BMOV is the bioactive species

The Conformational Universe of Proteins and Peptides: Tales of Order and Disorder

Proteins represent one of the most abundant classes of biological macromolecules and play crucial roles in a vast array of physiological and pathological processes. The knowledge of the 3D structure of a protein, as well as the possible conformational transitions occurring upon interaction with diverse ligands, are essential to fully comprehend its biological function.In addition to globular, well-folded proteins, over the past few years, intrinsically disordered proteins (IDPs) have received a lot of attention. IDPs are usually aggregation-prone and may form toxic amyloid fibers and oligomers associated with several human pathologies. Peptides are smaller in size than proteins but similarly represent key elements of cells. A few peptides are able to work as tumor markers and find applications in the diagnostic and therapeutic fields. The conformational analysis of bioactive peptides is important to design novel potential drugs acting as selective modulators of specific receptors or enzymes. Nevertheless, synthetic peptides reproducing different protein fragments have frequently been implemented as model systems in folding studies relying on structural investigations in water and/or other environments.This book contains contributions (seven original research articles and five reviews published in the journal Molecules) on the above-described topics and, in detail, it includes structural studies on globular folded proteins, IDPs and bioactive peptides. These works were conducted usingdifferent experimental methods

Physical activity and hypertension in South African adults

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.5 South Africa LicenseEstimates suggest that approximately 6-million South Africans have hypertension, with half classified as stage 1 (mild). Mindful of the cost of lifelong drug therapy, the South African Hypertension Society guidelines suggest delaying drug therapy through lifestyle modification (increased physical activity and weight management) in all but those with the highest risk. This pilot study examined the relationship of BP with physical activity and bodyweight in black South African adults employed in physical occupationsNon peer reviewe

A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Biochimica et Biophysica Acta - Biomembranes 1788 (2009): 1255-1262, doi:10.1016/j.bbamem.2008.12.016.Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na+/Ca2+ exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far- UV circular dichroism and infrared spectra suggest a majority of β-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten β-sheets and two short α- helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.The work was supported by Grants from the US National Science Foundation [MCB 0444598], Fondo Nacional para Investigaciones Científicas y Tecnológicas [PICT-05- 12397 and PICT-05-38073], Consejo Nacional de Investigfaciones Científicas y Técnicas [PIP 5118 and PIP 5593] Secretaría de Ciencia y Técnica Universidad Nacional de Córdoba, Argentina, Fondo Nacional para Ciencia y Técnica [S1-9900009046 and G- 2001000637] and Fundación Polar, Venezuela and The Rhode Island Idea Network of Biomedical Research Excellence (INBRE)

Cleavage of DNA by the Insulin-Mimetic Compound, NH4[VO(O2)2(phen)]

The kinetics and mechanism of cleavage of DNA by the insulin-mimetic peroxo-vanadate NH4[VO(O2)2(phen)], pV, are described. In the presence of low energy UV radiation or biologically common reducing agents, pV decomposes into the monomer, dimer, and tetramer of vanadate and an uncharacterized compound of V4+ as shown by 51V NMR, ESR, and absorption spectra. The rate of photodecomposition of pV is reduced in the presence of calf thymus DNA, indicating that a decomposition product of the peroxo-vanadate, that is important in the destruction pathway of the complex, is interacting with DNA. This species, probably a short-lived complex of V4+, may also be responsible for the observed catalytic decomposition of pV in the absence of DNA by ascorbate. If closed circular pBR322 DNA is present when the peroxo-vanadate is destroyed by either UV radiation or reducing agents, the polymer may have its sugar-phosphate backbone broken. Closed circular DNA (form I) is converted into nicked circular DNA (form II) and linear DNA (form III). The amounts of the various forms produced as a function of irradiation time and peroxo-vanadate concentration were fit to a kinetic model to derive rate constants for the conversions. The kinetic analysis shows that pV is a single-strand nicking agent which exhibits some base and/or sequence preference. Furthermore, the pH dependences of the rates for conversion of form I to form II and for conversion of form II to form III are different, indicating that the nature of the chemistry at the site of cleavage on DNA influences further cutting by activated pV. Reduced amounts of DNA breakage in the presence of various salts and metal binding ligands indicate that a short-lived reactive complex of V4+, not the V4+ species detected by ESR at long irradiation times, is important in the cleavage process. The susceptibility of pV to decomposition by biologically common reducing agents suggests that metabolites of the agent, and not the compound itself, are responsible for its insulin-mimetic effects

High Resolution Mass Spectrometry as a Platform for the Analysis of Polyoxometalates, their Solution Phase Dynamics, and their Biological Interactions.

Polyoxometalates (POMs) are a class of inorganic molecule of increasing interest to the inorganic, bioinorganic and catalytic communities among many others. While their prevalence in research has increased, tools and methodologies for the analysis of their fundamental characteristics still need further development. Decavanadate (V10) specifically has been postulated to have several unique properties that have not been confirmed independently. Mass spectrometry (MS) and its ability to determine the composition of solution phase species by both mass and charge is uniquely well suited to the analysis of POMs. In this work we utilized high-resolution mass spectrometry to characterize V10 in aqueous solution. We were able to observe a unique loss of water fragmentation pathway and prove the existence of partially reduced V10 species in solution. Due to the high negative charge of the V10 complex, it was also investigated as an analogue to heparin sulfate which has been shown to disrupt the infectivity cycle of SARS-CoV-2. Native mass spectrometry was used to demonstrate the ability of V10 to bind to the receptor binding domain of the spike protein. Additionally, the ability to disrupt the formation of a complex between the receptor binding domain (RBD) of the spike protein and the ectodomain of the angiotensin-converting enzyme 2 (ACE2) receptor was likewise investigated using native MS. This mechanism of action against SARS-CoV-2 was confirmed with cell viability studies. Further investigation of the solution phase dynamics of V10 was carried out by performing an oxygen exchange experiment coupled with high resolution mass spectrometry. The resulting exchange profiles revealed the existence of multiple populations of V10, including novel metastable conformations of V10. Further analysis of the oxygen exchange data yielded kinetic information about these metastable states and their role in the solution phase dynamics of V10. Finally, we also demonstrated the utility of bulk electrolysis in the synthesis of POV nanocages with unique ratios of reduced VIV centers. MS analysis of the species showed that the VO4 centered nanocage V19O46 is a precursor to the more stable chloride centered V15O36Cl which was shown to have unique solution phase dynamics upon subsequent oxygen exchange analysis. The work presented demonstrates the utility of mass spectrometry and isotopic exchange in the analysis of POMs

Proteomic analysis reveals the diversity and complexity of membrane proteins in chickpea (Cicer arietinum L.)

Background Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades. Results To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains. Conclusions The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses