Multicomponent Enzyme Complex Research Articles

Intersecting Stories of the Phagocyte NADPH Oxidase and Chronic Granulomatous Disease.

Neutrophils serve as the circulating cells that respond early and figure prominently in human host defense to infection and in inflammation in other settings. Optimal oxidant-dependent antimicrobial activity by neutrophils relies on the ability of stimulated phagocytes to utilize a multicomponent NADPH oxidase to generate oxidants. The frequent, severe, and often fatal infections experienced by individuals with chronic granulomatous disease (CGD), an inherited disorder in which one of the NADPH oxidase components is absent or dysfunctional, underscore the link between a functional phagocyte NADPH oxidase and robust host protection against microbial infection.The history of the discovery and characterization of the normal neutrophil NADPH oxidase and the saga of recognizing CGD and its underlying causes together illustrate how the observations of astute clinicians and imaginative basic scientists synergize to forge new understanding of both basic cell biology and pathogenesis of human disease.In this chapter, we review the events in the stepwise evolution of our understanding of the phagocyte NADPH oxidase, both in the context of normal human neutrophil function and in the setting of CGD. The phagocyte oxidase complex employs a heterodimeric transmembrane protein composed of gp91phox and p22phox to relay electrons from NADPH to molecular oxygen, while other cofactors contribute to localization and regulation of the activity of the assembled oxidase. The b-type cytochrome gp91phox, also known as NOX2, serves as the catalytic component of this multicomponent enzyme complex. Although many of the features of the composition and regulation of the phagocyte oxidase may apply as well to NOX2 expressed in non-phagocytes and to other members of the NOX protein family, exceptions exist and pose special challenges to investigators exploring the biology of NADPH oxidases.

Regulation of Ceramide Synthase by Casein Kinase 2-dependent Phosphorylation in Saccharomyces cerevisiae

Complex sphingolipids are important components of eukaryotic cell membranes and, together with their biosynthetic precursors, including sphingoid long chain bases and ceramides, have important signaling functions crucial for cell growth and survival. Ceramides are produced at the endoplasmic reticulum (ER) membrane by a multicomponent enzyme complex termed ceramide synthase (CerS). In budding yeast, this complex is composed of two catalytic subunits, Lac1 and Lag1, as well as an essential regulatory subunit, Lip1. Proper formation of ceramides by CerS has been shown previously to require the Cka2 subunit of casein kinase 2 (CK2), a ubiquitous enzyme with multiple cellular functions, but the precise mechanism involved has remained unidentified. Here we present evidence that Lac1 and Lag1 are direct targets for CK2 and that phosphorylation at conserved positions within the C-terminal cytoplasmic domain of each protein is required for optimal CerS activity. Our data suggest that phosphorylation of Lac1 and Lag1 is important for proper localization and distribution of CerS within the ER membrane and that phosphorylation of these sites is functionally linked to the COP I-dependent C-terminal dilysine ER retrieval pathway. Together, our data identify CK2 as an important regulator of sphingolipid metabolism, and additionally, because both ceramides and CK2 have been implicated in the regulation of cancer, our findings may lead to an enhanced understanding of their relationship in health and disease.

A pH-Dependent Kinetic Model of Dihydrolipoamide Dehydrogenase from Multiple Organisms

Dihydrolipoamide dehydrogenase is a flavoenzyme that reversibly catalyzes the oxidation of reduced lipoyl substrates with the reduction of NAD+ to NADH. In vivo, the dihydrolipoamide dehydrogenase component (E3) is associated with the pyruvate, α-ketoglutarate, and glycine dehydrogenase complexes. The pyruvate dehydrogenase (PDH) complex connects the glycolytic flux to the tricarboxylic acid cycle and is central to the regulation of primary metabolism. Regulation of PDH via regulation of the E3 component by the NAD+/NADH ratio represents one of the important physiological control mechanisms of PDH activity. Furthermore, previous experiments with the isolated E3 component have demonstrated the importance of pH in dictating NAD+/NADH ratio effects on enzymatic activity. Here, we show that a three-state mechanism that represents the major redox states of the enzyme and includes a detailed representation of the active-site chemistry constrained by both equilibrium and thermodynamic loop constraints can be used to model regulatory NAD+/NADH ratio and pH effects demonstrated in progress-curve and initial-velocity data sets from rat, human, Escherichia coli, and spinach enzymes. Global fitting of the model provides stable predictions to the steady-state distributions of enzyme redox states as a function of lipoamide/dihydrolipoamide, NAD+/NADH, and pH. These distributions were calculated using physiological NAD+/NADH ratios representative of the diverse organismal sources of E3 analyzed in this study. This mechanistically detailed, thermodynamically constrained, pH-dependent model of E3 provides a stable platform on which to accurately model multicomponent enzyme complexes that implement E3 from a variety of organisms.

Ethanol‐induced ROS production is prevented by globular adiponectin via modulation of Nox2 expression in macrophages (844.19)

Adiponectin predominantly secreted from adipose tissue plays a crucial role in lipid metabolism and possesses potent anti‐inflammatory properties. Adipoonectin has been shown to generate protective effects in a number of pathophysiological states. Recently, it has been shown that adiponectin prevents ROS production induced by stimulation with various stimuli. In the present study, we investigated the effect of globular adiponectin (gAcrp) on ethanol‐induced ROS production in macrophages and underlying mechanisms. Herein, we found that gAcrp efficiently suppresses ethanol‐induced ROS production both in RAW 264.7 macrophages and murine peritoneal macrophages. We next investigated the role of NADPH oxidase, comprising a multi‐component enzyme complex, in the suppression of ROS production by gAcrp, since NADPH oxidase plays a critical role in ethanol‐induced ROS production in macrophages. Globular adiponectin prevented ethanol‐induced NAPDH oxidase activation. Particularly, it inhibited ethanol‐induced increase in expression of Nox2 and p22phox. Furthermore, gAcrp restored ethanol‐induced activation of p38MAPK‐NF‐κB pathway, which is critical for Nox2 expression. Taken together, these results suggest that adiponectin prevents ethanol‐induced ROS production via modulation of NADPH oxidase in macrophages.

Role of NADPH Oxidases in Liver Fibrosis

Hepatic fibrosis is the common pathophysiologic process resulting from chronic liver injury, characterized by the accumulation of an excessive extracellular matrix. Multiple lines of evidence indicate that oxidative stress plays a pivotal role in the pathogenesis of liver fibrosis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. In addition to phagocytic NOX2, there are six nonphagocytic NOX proteins. In the liver, NOX is functionally expressed both in the phagocytic form and in the nonphagocytic form. NOX-derived ROS contributes to various kinds of liver disease caused by alcohol, hepatitis C virus, and toxic bile acids. Recent evidence indicates that both phagocytic NOX2 and nonphagocytic NOX isoforms, including NOX1 and NOX4, mediate distinct profibrogenic actions in hepatic stellate cells, the main fibrogenic cell type in the liver. The critical role of NOX in hepatic fibrogenesis provides a rationale to assess pharmacological NOX inhibitors that treat hepatic fibrosis in patients with chronic liver disease. Although there is compelling evidence indicating a crucial role for NOX-mediated ROS generation in hepatic fibrogenesis, little is known about the expression, subcellular localization, regulation, and redox signaling of NOX isoforms in specific cell types in the liver. Moreover, the exact mechanism of NOX-mediated fibrogenic signaling is still largely unknown. A better understanding through further research about NOX-mediated fibrogenic signaling may enable the development of novel anti-fibrotic therapy using NOX inhibition strategy. Antio

Preliminary X-ray diffraction study of crystals of photosystem II from Thermosynechococcus elongates

Photosystem II (PSII) is a multicomponent enzyme complex that catalyzes the light-induced water splitting to molecular oxygen, protons, and electrons. Photosystem II is located in the membranes of cyanobacteria, green algae, and plants. The crystallization of this complex from the thylakoid membranes poses great difficulties. The high sensitivity of photosystem II to light and radiation has an adverse effect on the crystal quality, as well as on the quality of X-ray diffraction data. This is the reason why the crystal structure of PSII from Thermosynechococcus elongates has as yet not been determined at high resolution. The optimization of the strategy for collecting X-ray diffraction data from PSII crystals has resulted in an increase in the resolution to 2.75 A, which made it possible to determine the positions of ions and some water molecules playing an important role in the functioning of PSII.

A Noncellulosomal Mannanase26E Contains a CBM59 inClostridium cellulovorans

A multicomponent enzyme-complex prevents efficient degradation of the plant cell wall for biorefinery. In this study, the method of identifying glycoside hydrolases (GHs) to degrade hemicelluloses was demonstrated. The competence of C. cellulovorans, which changes to be suitable for degradation of each carbon source, was used for the method. C. cellulovorans was cultivated into locust bean gum (LBG) that is composed of galactomannan. The proteins produced by C. cellulovorans were separated into either fractions binding to crystalline cellulose or not. Proteins obtained from each fraction were further separated by SDS-PAGE and were stained with Coomassie Brilliant Blue and were detected for mannanase activity. The proteins having the enzymatic activity for LBG were cut out and were identified by mass spectrometry. As a result, four protein bands were classified into glycosyl hydrolase family 26 (GH26) mannanases. One of the identified mannanases, Man26E, contains a carbohydrate-binding module (CBM) family 59, which binds to xylan, mannan, and Avicel. Although mannose and galactose are the same as a hexose, the expression patterns of the proteins from C. cellulovorans were quite different. More interestingly, zymogram for mannanase activity showed that Man26E was detected in only LBG medium.

Emerging role of oxidative stress in metabolic syndrome and cardiovascular diseases: important role of Rac/NADPH oxidase

'Oxidative stress' is a term defining states of elevated reactive oxygen species (ROS) levels. Normally, ROS control several physiological processes, such as host defence, biosynthesis of hormones, fertilization and cellular signalling. However, oxidative stress has been involved in different pathologies, including metabolic syndrome and numerous cardiovascular diseases. A major source of ROS involved in both metabolic syndrome and cardiovascular pathophysiology is the NADPH oxidase (NOX) family of enzymes. NOX is a multi-component enzyme complex that consists of membrane-bound cytochrome b-558, which is a heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox and p67phox, and the small GTP-binding protein Rac1. Rac1 plays many important biological functions in cells, but perhaps the most unique function of Rac1 is its ability to bind and activate the NOX complex. Furthermore, Rac1 has been reported to be a key regulator of oxidative stress through its co-regulatory effects on both nitric oxide (NO) synthase and NOX. Therefore, the main goal of this review is to give a brief outline about the important role of the Rac1-NOX axis in the pathophysiology of both metabolic syndrome and cardiovascular disease.

Two Pfam protein families characterized by a crystal structure of protein lpg2210 from Legionella pneumophila

BackgroundEvery genome contains a large number of uncharacterized proteins that may encode entirely novel biological systems. Many of these uncharacterized proteins fall into related sequence families. By applying sequence and structural analysis we hope to provide insight into novel biology.ResultsWe analyze a previously uncharacterized Pfam protein family called DUF4424 [Pfam:PF14415]. The recently solved three-dimensional structure of the protein lpg2210 from Legionella pneumophila provides the first structural information pertaining to this family. This protein additionally includes the first representative structure of another Pfam family called the YARHG domain [Pfam:PF13308]. The Pfam family DUF4424 adopts a 19-stranded beta-sandwich fold that shows similarity to the N-terminal domain of leukotriene A-4 hydrolase. The YARHG domain forms an all-helical domain at the C-terminus. Structure analysis allows us to recognize distant similarities between the DUF4424 domain and individual domains of M1 aminopeptidases and tricorn proteases, which form massive proteasome-like capsids in both archaea and bacteria.ConclusionsBased on our analyses we hypothesize that the DUF4424 domain may have a role in forming large, multi-component enzyme complexes. We suggest that the YARGH domain may play a role in binding a moiety in proximity with peptidoglycan, such as a hydrophobic outer membrane lipid or lipopolysaccharide.

NADPH oxidase mediated oxidative stress in hepatic fibrogenesis

NADPH oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. ROS is involved as key secondary messengers in numerous signaling pathways, and NADPH oxidases complex has been increasingly recognized as key elements of intracellular signaling of hepatic fibrogenesis. In the liver, NADPH oxidase is functionally expressed both in the phagocytic form and in the non-phagocytic form. The non-phagocytic NADPH oxidase complex is structurally and functionally similar to the phagocytic NADPH, resulting in reduction of molecular oxygen to generate superoxide. There are six homologous NOX proteins in the non-phagocytic forms of NADPH oxidase. An emerging concept is that both phagocytic and nonphagocytic NADPH oxidase components in hepatic stellate cells (HSCs) mediate hepatic fibrosis, suggesting its potential role as a pharmacological target for anti-fibrotic therapy. The molecular components and signaling pathways of various NADPH oxidase homologues that are critical for the fibrotic activity in HSCs need to be more clearly identified.

Structural consequences of effector protein complex formation in a diiron hydroxylase.

Carboxylate-bridged diiron hydroxylases are multicomponent enzyme complexes responsible for the catabolism of a wide range of hydrocarbons and as such have drawn attention for their mechanism of action and potential uses in bioremediation and enzymatic synthesis. These enzyme complexes use a small molecular weight effector protein to modulate the function of the hydroxylase. However, the origin of these functional changes is poorly understood. Here, we report the structures of the biologically relevant effector protein-hydroxylase complex of toluene 4-monooxygenase in 2 redox states. The structures reveal a number of coordinated changes that occur up to 25 A from the active site and poise the diiron center for catalysis. The results provide a structural basis for the changes observed in a number of the measurable properties associated with effector protein binding. This description provides insight into the functional role of effector protein binding in all carboxylate-bridged diiron hydroxylases.

Downregulation of p22phox in Retinal Pigment Epithelial Cells Inhibits Choroidal Neovascularization in Mice

Choroidal neovascularization (CNV) occurs in a variety of chorioretinal diseases including age-related macular degeneration (AMD), and is the major cause of severe visual loss in patients with AMD. Oxidative stress has been thought to play an important role in the development of CNV. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the major intracellular sources of reactive oxygen species (ROS) in the vascular system. In this study, we examined the expression of p22phox, an integral subunit in the NADPH oxidase complex, in the mouse eye. We determined that p22phox is expressed in the retinal pigment epithelial (RPE) cells and inner retinal neurons. A small-interfering RNA (siRNA) designed against p22phox efficiently reduced the expression of the protein in the eye when delivered by means of recombinant adeno-associated virus (AAV) vector. Vector treatment inhibited CNV in the mouse when delivered into the subretinal space where RPE cells were transduced. These results suggest that NADPH oxidase-mediated ROS production in RPE cells may play an important role in the pathogenesis of neovascular AMD, and that this pathway may represent a new target for therapeutic intervention in AMD.

P47phox-deficient immune microenvironment signals dysregulate naive T-cell apoptosis

The phagocyte NADPH oxidase is a multicomponent enzyme complex mediating microbial killing. We find that NADPH oxidase p47(phox)-deficient (p47(phox-/-)) chronic granulomatous disease (CGD) mice develop lymph node hyperplasia even without obvious infection, where increased number of T and B lymphocytes is associated with increased percent of naïve cells and a lower T : B cell ratio than wild type. Paradoxically, despite lymphoid hyperplasia in vivo, when lymphocytes are placed in culture, p47(phox-/-) CD8(+) lymphocytes progress more rapidly to apoptosis than wild type. This is associated in cultured p47(phox-/-) CD8(+) lymphocytes with the induction of proapoptotic Bim and Puma expression, increased mitochondrial outer membrane permeabilization and depressed Bcl-2 expression. Addition of IL-7 to the culture partially corrects Bcl-2 levels in cultured p47(phox-/-) CD8(+) lymphocytes and improves the survival. Adding glucose oxidase to the culture to generate hydrogen peroxide along with IL-7 further improves p47(phox-/-) CD8(+) lymphocyte survival, but only to 30% of wild type. We conclude that p47(phox-/-) CD8(+) lymphocytes have an intrinsic survival defect likely in part related to the oxidase deficiency, but in vivo in lymph nodes of CGD mice, there are microenvironmental factors yet to be delineated that suppress the progression of apoptosis and allow the accumulation of lymphocytes leading to lymphoid hyperplasia.

Physiologie der Blutgerinnung und Fibrinolyse

The principles of initiator and amplifications reactions of blood coagulation and fibrinolysis will be presented and discussed in relation to various regulatory pathways of haemostasis. In particular, cell surface-dependent activation and inhibition reactions are characteristics of multicomponent enzyme complexes that also allow the endogenous control of the haemostasis system. The understanding of these relationships in complications of haemostasis has lead to different strategies for the therapeutic intervention with pro- and anticoagulant substances.

The microglial NADPH oxidase complex as a source of oxidative stress in Alzheimer's disease

Alzheimer's disease is the most common cause of dementia in the elderly, and manifests as progressive cognitive decline and profound neuronal loss. The principal neuropathological hallmarks of Alzheimer's disease are the senile plaques and the neurofibrillary tangles. The senile plaques are surrounded by activated microglia, which are largely responsible for the proinflammatory environment within the diseased brain. Microglia are the resident innate immune cells in the brain. In response to contact with fibrillar beta-amyloid, microglia secrete a diverse array of proinflammatory molecules. Evidence suggests that oxidative stress emanating from activated microglia contribute to the neuronal loss characteristic of this disease. The source of fibrillar beta-amyloid induced reactive oxygen species is primarily the microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The NADPH oxidase is a multicomponent enzyme complex that, upon activation, produces the highly reactive free radical superoxide. The cascade of intracellular signaling events leading to NADPH oxidase assembly and the subsequent release of superoxide in fibrillar beta-amyloid stimulated microglia has recently been elucidated. The induction of reactive oxygen species, as well as nitric oxide, from activated microglia can enhance the production of more potent free radicals such as peroxynitrite. The formation of peroxynitrite causes protein oxidation, lipid peroxidation and DNA damage, which ultimately lead to neuronal cell death. The elimination of beta-amyloid-induced oxidative damage through the inhibition of the NADPH oxidase represents an attractive therapeutic target for the treatment of Alzheimer's disease.

Crystal Structure of Lipoate-Protein Ligase A Bound with the Activated Intermediate: INSIGHTS INTO INTERACTION WITH LIPOYL DOMAINS

Lipoic acid is the covalently attached cofactor of several multi-component enzyme complexes that catalyze key metabolic reactions. Attachment of lipoic acid to the lipoyl-dependent enzymes is catalyzed by lipoate-protein ligases (LPLs). In Escherichia coli, two distinct enzymes lipoate-protein ligase A (LplA) and lipB-encoded lipoyltransferase (LipB) catalyze independent pathways for lipoylation of the target proteins. The reaction catalyzed by LplA occurs in two steps. First, LplA activates exogenously supplied lipoic acid at the expense of ATP to lipoyl-AMP. Next, it transfers the enzyme-bound lipoyl-AMP to the epsilon-amino group of a specific lysine residue of the lipoyl domain to give an amide linkage. To gain insight into the mechanism of action by LplA, we have determined the crystal structure of Thermoplasma acidophilum LplA in three forms: (i) the apo form; (ii) the ATP complex; and (iii) the lipoyl-AMP complex. The overall fold of LplA bears some resemblance to that of the biotinyl protein ligase module of the E. coli biotin holoenzyme synthetase/bio repressor (BirA). Lipoyl-AMP is bound deeply in the bifurcated pocket of LplA and adopts a U-shaped conformation. Only the phosphate group and part of the ribose sugar of lipoyl-AMP are accessible from the bulk solvent through a tunnel-like passage, whereas the rest of the activated intermediate is completely buried inside the active site pocket. This first view of the activated intermediate bound to LplA allowed us to propose a model of the complexes between Ta LplA and lipoyl domains, thus shedding light on the target protein/lysine residue specificity of LplA.

PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites

tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.

Lysosomal Acid α-Glucosidase Consists of Four Different Peptides Processed from a Single Chain Precursor

Pompe's disease is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and N-glycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78-113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204-782) contains the 3.9- and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.

Selected Non-steroidal Anti-inflammatory Drugs and Their Derivatives Target γ-Secretase at a Novel Site: EVIDENCE FOR AN ALLOSTERIC MECHANISM

Gamma-secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-beta (Abeta) peptides from processing intermediates of the beta-amyloid precursor protein. Because Abeta peptides are thought to be causative for Alzheimer's disease, inhibiting gamma-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of gamma-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic Abeta(1-42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of Abeta(1-42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective gamma-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the gamma-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of gamma (42)-cleavage over alternative cleavages in the beta-amyloid precursor protein and, more importantly, alternative substrates.

Biochemistry and physiology of blood coagulation and fibrinolysis

The principles of initiator and amplification reactions of blood coagulation and fibrinolysis will be presented and discussed in relation to various regulatory pathways of haemostasis. In particular, cell surface-dependent activation and inhibition reactions are characteristics of multi-component enzyme complexes that also allow the endogenous physiological control of the haemostasis system. The understanding of these relationships in complications of haemostasis has led to different available strategies for the therapeutic intervention with pro- and anticoagulant substances.