Arto Annila

Sphingomyelinase Induces Aggregation and Fusion, but Phospholipase A2 Only Aggregation, of Low Density Lipoprotein (LDL) Particles TWO DISTINCT MECHANISMS LEADING TO INCREASED BINDING STRENGTH OF LDL TO HUMAN AORTIC PROTEOGLYCANS

During atherogenesis, low density lipoprotein (LDL) particles bind to extracellular matrix proteoglycans in the arterial wall, become modified, and appear as aggregated and fused particles. Sphingomyelinase (SMase) and phospholipase A2 (PLA2) have been found in the arterial wall, and, moreover, lesional LDL shows signs of hydrolysis of both sphingomyelin and phosphatidylcholine. We have now studied the effects of these two lipolytic modifications on the aggregation and fusion of LDL particles by hydrolyzing the particles with Bacillus cereus SMase or bee venom PLA2. In addition, the binding strengths of the modified LDL to human aortic proteoglycans (PG) were analyzed on an affinity column. We found that SMase induced aggregation and fusion of LDL, but PLA2 induced only aggregation of the particles. In addition, the SMase-induced aggregation and fusion of LDL was promoted by pretreatment of LDL with PLA2. Determination of the binding strengths of the hydrolyzed LDL revealed that mere lipolysis of LDL without aggregation or fusion, either by SMase or PLA2, did not affect the binding of the particles to PG. Aggregation and fusion of lipolyzed LDL particles, however, increased their strength of binding to PG. Active lysine residues in apolipoprotein B-100 (apoB-100) appear to be involved in the binding of LDL to PG, and, in fact, quantitative13C NMR analysis revealed that, in the fused LDL particles, the number of active lysine residues per apoB-100 moiety was increased. Moreover, aggregation and fusion of LDL increased the number of apoB-100 copies and, consequently, the number of active lysine residues per aggregate or fused particle. Our present findings therefore (i) show that treatment of LDL with SMase and PLA2 generates modified LDL particles, which then bind to human aortic PG with increased strength, and (ii) suggest that SMase- and PLA2-induced aggregation and fusion of LDL are potential mechanisms leading to focal retention of extracellular lipid in the arterial wall.

Binding of Levosimendan, a Calcium Sensitizer, to Cardiac Troponin C

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.

Natural selection for least action

The second law of thermodynamics is a powerful imperative that has acquired several expressions during the past centuries. Connections between two of its most prominent forms, i.e. the evolutionary principle by natural selection and the principle of least action, are examined. Although no fundamentally new findings are provided, it is illuminating to see how the two principles rationalizing natural motions reconcile to one law. The second law, when written as a differential equation of motion, describes evolution along the steepest descents in energy and, when it is given in its integral form, the motion is pictured to take place along the shortest paths in energy. In general, evolution is a non-Euclidian energy density landscape in flattening motion.

Oxidation of Low Density Lipoprotein Particles Decreases Their Ability to Bind to Human Aortic Proteoglycans DEPENDENCE ON OXIDATIVE MODIFICATION OF THE LYSINE RESIDUES

Oxidation of low density lipoprotein (LDL) leads to its rapid uptake by macrophages in vitro, but no detailed studies have addressed the effect of oxidation on the binding of LDL to proteoglycans. We therefore treated LDL with various substances: copper sulfate, 2,2′-azobis(2-amidinopropane)hydrochloride (AAPH), soybean lipoxygenase, and mouse peritoneal macrophages, and determined the extent to which the oxidatively modified LDL bound to human aortic proteoglycans in an affinity column. Oxidation of LDL with copper, AAPH, or macrophages, all of which increased its electrophoretic mobility, was associated with reduced binding to proteoglycans, until strongly oxidized LDL was totally unable to bind to them. After treatment of LDL with soybean lipoxygenase, the change in electrophoretic mobility was small, and the amount of binding to proteoglycans was only slightly decreased. The increased electrophoretic mobility of oxidized LDL reflects modification of the lysine residues of apolipoprotein B-100 (apoB-100). To mimic the oxidative modification of lysines, we treated LDL with malondialdehyde. This treatment also totally prevented the binding of LDL to proteoglycans. In contrast, if the lysine residues of apoB-100 were methylated to shield them against oxidative modification, subsequent treatment of LDL with copper sulfate failed to reduce the degree of LDL binding to proteoglycans. Finally, the active lysine residues in the oxidized LDL particles, which are thought to be involved in this binding, were quantified with NMR spectroscopy. In oxidized LDL, the number of these residues was found to be decreased. The present results show that, after modification of the lysine residues of apoB-100 during oxidation, the binding of LDL to proteoglycans is decreased, and suggest that oxidation of LDL tends to lead to intracellular rather than extracellular accumulation of LDL during atherogenesis.

Recognition of protein folds via dipolar couplings

Alignment of proteins in dilute liquid crystalline medium gives rise to residual dipolar couplings which provide orientational information of vectors connecting the interacting nuclei. Considering that proteins are mainly composed of regular secondary structures in a finite number of different mutual orientations, main chain dipolar couplings appear sufficient to reveal structural resemblance. Similarity between dipolar couplings measured from a protein and corresponding values computed from a known structure imply homologous structures. For dissimilar structures the agreement between experimental and calculated dipolar couplings remains poor. In this way protein folds can be readily recognized prior to a comprehensive structure determination. This approach has been demonstrated by showing the similarity in fold between the hitherto unknown structure of calerythrin and sarcoplasmic calcium-binding proteins from Nereis diversicolor and Branchiostoma lanceolatum with known crystal structures.

Physical foundations of evolutionary theory

Abstract The theory of evolution by natural selection is herein subsumed by the 2nd law of thermodynamics. The mathematical form of evolutionary theory is based on a re-examination of the probability concept that underlies statistical physics. Probability regarded as physical must include, in addition to isoenergic combinatorial configurations, also energy in conditional circumstances. Consequently, entropy as an additive logarithmic probability measure is found to be a function of the free energy, and the process toward the maximum entropy state is found equivalent to evolution toward the free energy minimum in accordance with the basic maxim of chemical thermodynamics. The principle of increasing entropy when given as an equation of motion reveals that expansion, proliferation, differentiation, diversification, and catalysis are all ways for a system to evolve toward the stationary state in its respective surroundings. Intriguingly, the equation of evolution cannot be solved when there remain degrees of freedom to consume the free energy, and hence evolutionary trajectories of a non-Hamiltonian system remain intractable. Finally, when to-and-from flows of energy are balanced between a system and its surroundings, the system is at the Lyapunov-stable stationary state. The principle of maximal energy dispersal, equivalent to the maximal rate of entropy production, gives rise to the ubiquitous characteristics, conventions, and regularities found in nature, where thermodynamics makes no demarcation line between animate and inanimate.

All in Action

Abstract: The principle of least action provides a holistic worldview in which Nature in its entirety and every detail is described in terms of actions. Each and every action is ultimately composed of one or multiple of the most elementary actions which relates to Planck’s constant. Elements of space are closed actions, known as fermions, whereas elements of time are open actions, known as bosons. The actions span an energy landscape, the Universe, which evolves irreversibly according to the 2nd law of thermodynamics by diminishing energy density differences in least time. During evolution densely-curled actions unfold step-by-step when opening up and expelling one or multiple elementary actions to their surrounding sparser space. The energy landscape will process from one symmetry group to another until the equivalence to its dual, i.e. , the surrounding density has been attained. The scale-free physical portrayal of nature in terms of actions does not recognize any fundamental difference between fundamental particles and fundamental forces. Instead a plethora of particles and a diaspora of forces are perceived merely as diverse manifestations of a natural selection for various mechanisms and ways to decrease free energy in the least time.

Natural patterns of energy dispersal

Universal patterns such as power-law dependences, skewed distributions, tree-like structures, networks and spirals are associated with energy dispersal processes using the principle of least action. Also ubiquitous temporal courses such as sigmoid growth, bifurcations and chaos are ascribed to the decrease of free energy in the least time. Moreover, emergence of natural standards such as the common genetic code and chirality consensus of amino acids are understood to follow from the quest to maximize the dispersal of energy. Many mathematical functions that model natural patterns and processes are found as approximations of the evolutionary equation of motion that has been derived from statistical physics of open systems. The evolutionary processes can be described as flows of energy that run from high energy sources to low energy sinks in the least time. However, the equation of evolution cannot be solved in general because the flows of energy and their driving forces are inseparable. Since the energy of the system keeps changing, the paths of evolution cannot be integrated from a given initial state to a final state. Although evolutionary courses of these non-Hamiltonian systems with two or more alternative ways of dissipation cannot be predicted, the flows of energy will search and naturally select paths of least action, known as geodesics, to consume free energy in the least time. The scale-invariant natural patterns follow from this natural law that impinges on processes at all scales of space and time.

Solution Structure of Nodularin AN INHIBITOR OF SERINE/THREONINE-SPECIFIC PROTEIN PHOSPHATASES

The three-dimensional solution structure of nodularin was studied by NMR and molecular dynamics simulations. The conformation in water was determined from the distance and dihedral data by distance geometry and refined by iterative relaxation matrix analysis. The cyclic backbone adopts a well defined conformation but the remote parts of the side chains of arginine as well as the amino acid derivative Adda have a large spatial dispersion. For the unusual amino acids the partial charges were calculated and nodularin was subjected to molecular dynamic simulations in water. A good agreement was found between experimental and computational data with hydrogen bonds, solvent accessibility, molecular motion, and conformational exchange. The three-dimensional structure resembles very closely that of microcystin-LR in the chemically equivalent segment. Therefore, it is expected that the binding of both microcystins and nodularins to serine/threonine-specific protein phosphatases is similar on an atomic level.

Economies evolve by energy dispersal

Abstract: Economic activity can be regarded as an evolutionary process governed by the 2 nd law of thermodynamics. The universal law, when formulated locally as an equation of motion, reveals that a growing economy develops functional machinery and organizes hierarchically in such a way as to tend to equalize energy density differences within the economy and in respect to the surroundings it is open to. Diverse economic activities result in flows of energy that will preferentially channel along the most steeply descending paths, leveling a non-Euclidean free energy landscape. This principle of ‗maximal energy dispersal‘, equivalent to the maximal rate of entropy production, gives rise to economic laws and regularities. The law of diminishing returns follows from the diminishing free energy while the relation between supply and demand displays a quest for a balance among interdependent energy densities. Economic evolution is dissipative motion where the driving forces and energy flows are inseparable from each other. When there are multiple degrees of freedom, economic growth and decline are inherently impossible to forecast in detail. Namely, trajectories of an evolving economy are non-integrable,