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Monoglyceride lipase catalyzes the last step in the hydrolysis of stored triglycerides in the adipocyte and presumably also complements the action of lipoprotein lipase in degrading triglycerides from chylomicrons and very low density lipoproteins. Monoglyceride lipase was cloned from a mouse adipocyte cDNA library. The predicted amino acid sequence consisted of 302 amino acids, corresponding to a molecular weight of 33,218. The sequence showed no extensive homology to other known mammalian proteins, but a number of microbial proteins, including two bacterial lysophospholipases and a family of haloperoxidases, were found to be distantly related to this enzyme. By means of multiple sequence alignment and secondary structure prediction, the structural elements in monoglyceride lipase, as well as the putative catalytic triad, were identified. The residues of the proposed triad, Ser-122, in a GXSXG motif, Asp-239, and His-269, were confirmed by site-directed mutagenesis experiments. Northern blot analysis revealed that monoglyceride lipase is ubiquitously expressed among tissues, with a transcript size of about 4 kilobases.The sequential hydrolysis of stored triglycerides in adipose tissue is the result of a combined action of two lipases, hormone-sensitive lipase and monoglyceride lipase (MGL 1 ; EC 3.1.1.23). Hormone-sensitive lipase catalyzes the first and ratelimiting step, the hydrolysis of triglycerides, and also the subsequent hydrolysis of di-and monoglycerides (1). Hormonesensitive lipase has a marked, although not absolute, preference for the primary ester bond of glyceride substrates. It has been shown that MGL is required to obtain a complete degradation of monoglycerides to fatty acids and glycerol, i.e. in the absence of MGL there is an accumulation of monoglycerides (mainly 2-monoglycerides) (2). The main physiological role for MGL is probably to assure complete hydrolysis of monoglycerides formed during the lipolysis of stored triglycerides of the adipocyte. Another role for the enzyme could be to catalyze the hydrolysis of 2-monoglycerides formed as a result of lipoprotein lipase-catalyzed hydrolysis of triglycerides from chylomicrons and very low density lipoproteins. Lipoprotein lipase has monoglyceride-hydrolyzing activity, with an absolute preference for the primary ester bond (3). This lipase could therefore catalyze the hydrolysis of 1(3)-monoglycerides, which are formed through isomerization from 2-monoglycerides. However, since the rate of isomerization at pH 7.4 is low, it is more likely that a substantial fraction of the 2-monoglycerides, formed through the action of lipoprotein lipase, is transported into the adipocyte and hydrolyzed by MGL (4). It should be pointed out that besides these two enzymes, there is no evidence for any other monoglyceride-hydrolyzing activity of adipose tissue.MGL has been extensively purified from rat adipose tissue in our laboratory (5). The limited amounts of purified enzyme obtained have been used to study some of its enzymological and biochemical propertie...

Cloning and sequencing of the complementary DNA for platelet-derived endothelial cell growth factor indicates that it is a novel factor distinct from previously characterized proteins. The factor, a protein with a relative molecular mass of about 45,000, stimulates endothelial cell growth and chemotaxis in vitro and angiogenesis in vivo.

The Alzhlmer Af3 amyloid peptide (A3) is the principal protelnaceous component of amylold associated with Alzheimer disease (AD Since slight differences in structure may affect amyloidogenesis, a thorough knowledge of the actual AP composition of amyloid is therefore important. Moreover, knowledge of the exact structure is important for characterization of the proteolytic enzymes involved in AB formation. In addition, amyloid associated with normal aging has, to our knowledge, not been characterized biochemically and its peptide composition is unknown.In the present work, AP was purified from the cerebral cortex of a number of sporadic AD cases and nondemented elderly controls, as well as two familial AD (FAD) cases. One of the two FAD cases had the APP K670N/M671L mutation (13), and the other had the APP V717I mutation (14). Primary structures and relative abundances of the purified AB variants were determined by N-terminal microsequencing and electrospray-ionization mass spectrometry (ESI-MS).Alzheimer disease (AD) is associated with deposition of amyloid in the brain parenchyma and within the cerebromeningeal vasculature (for review, see ref. 1). Amyloid displaying properties similar to those of AD amyloid can also be detected in normal aging (2). Whether this amyloid accompanies normal aging or is an early histopathological sign of presymptomatic AD is not known. The AD-associated amyloid deposits are mainly composed of the 4-kDa Alzheimer A, amyloid peptide (AB) (3, 4). AB is a proteolytic fragment of a transmembrane glycoprotein, the Alzheimer AP amyloid precursor protein (APP) (5).Since the initial isolation of AP from amyloid deposits (3), a variety of methods for purification and analysis of the peptide have been used (4, 6-8). Various forms of the native peptide have been reported. For instance, it has been stated that the N terminus of AP is blocked (6), that AB is deposited as a mixture of N-terminally truncated ("ragged") variants (4), and that the C terminus is different in vascular and parenchymal AP (7). More recently, it was proposed that A/-(1-40) is the major variant in brain (9) and that cerebrovascular amyloid is composed primarily of A(-(1-40) and Af-(1-42) (10) (Fig. 1)

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Smad proteins regulate gene expression in response to TGFbeta signaling. Here we present evidence that Smad7 interacts with the transcriptional coactivator p300, resulting in acetylation of Smad7 on two lysine residues in its N terminus. Acetylation or mutation of these lysine residues stabilizes Smad7 and protects it from TGFbeta-induced degradation. Furthermore, we demonstrate that the acetylated residues in Smad7 also are targeted by ubiquitination and that acetylation of these lysine residues prevents subsequent ubiquitination. Specifically, acetylation of Smad7 protects it against ubiquitination and degradation mediated by the ubiquitin ligase Smurf1. Thus, our data suggest that competition between ubiquitination and acetylation of overlapping lysine residues constitutes a novel mechanism to regulate protein stability.

The pathological fibrillar deposits found in the heart and other organs of patients with senile systemic amyloidosis (SSA) and Swedish familial amyloidotic polyneuropathy (FAP) contain wild-type (wt) and a mutant form of transthyretin (TTR), respectively. Previously, it was reported that these two forms of amyloid have different molecular features and it was thus postulated that the mechanism responsible for TTR fibrillogenesis in SSA and FAP may differ. To document further the nature of the amyloid in these entities, detailed morphological, histochemical, immunological, and structural analyses of specimens obtained from 14 individuals with SSA and 11 Swedish FAP patients have been performed. Two distinct patterns of amyloid deposition (designated A and B) were evident. In pattern A, found in all SSA and five of 11 FAP cases, the amyloid had a homogeneous but patchy distribution within the sub-endocardium, sub-epicardium, and myocardium; exhibited weak congophilia and green birefringence; and was composed of tightly packed, short, unorientated fibrils. This material contained mainly approximately 79-residue C-terminal fragments of the amyloidogenic precursor protein. In pattern B, seen in the six other FAP patients, the amyloid appeared as thin streaks throughout the cardiac tissue; often surrounded individual muscle cells; was strongly congophilic and birefringent; had long fibrils arranged in parallel bundles, often penetrating into myocytes; and was composed of virtually intact TTR molecules. These findings provide substantive evidence for the morphological and structural heterogeneity of TTR fibrils and suggest that the two types of deposition may reflect fundamental differences in the pathogenesis of the TTR-associated amyloidoses.

A genetic approach is described to clarify the IgG-binding properties of the N-terminal portion of staphylococcal protein A (region E). Several gene fragments, encoding region E or B or protein A, have been cloned and expressed in Escherichia coli. The gene products were purified by IgG-affinity chromatography and subjected to structural and functional analyses. Both fragments can be efficiently purified using this method, suggesting that region B as well as region E has Fc-binding activity. In addition, gene fusions were assembled giving fragments EB and EE, which both showed a divalent Fc-binding. These results demonstrate that protein A consists of five IgG-binding domains. The implications of these findings for the structure of protein-Aimmunoglobulin-G complexes are discussed.Staphylococcal protein A (SpA) plays an important role in molecular biology owing to its specific interaction with the Fc portion of immunoglobulins from many mammals [l -31. Biological responses to SpA include activation of the complement system, hypersensitivity reactions, cell-mediated cytotoxicity, interferon production, activation of polyclonal antibody synthesis and mitogenic stimulation of lymphocytes [4 -81. Many immunological methods have been developed and refined using SpA as a reagent, including immunoprecipitation techniques and double sandwich immunoassays [9, 101. In addition, solid-phase protein A has been used therapeutically to decrease the amount of circulating immunocomplexes in sera [l 13. Treatments involving continuous-flow extracorporal systems have been tried on patients with advanced carcinomas and acquired immunodeficient syndrome (AIDS).Because of its importance as an immunological tool, extensive structural and biochemical studies of the protein A molecule have been performed during the last two decades [12 -181. Fragmentation of the SpA molecule by trypsin digestion followed by purification and amino acids sequence analysis of the fragments suggested a tetrameric structure of the IgG-binding part [14, 151. A model was therefore proposed where SpA consists of four highly homologous Fc-binding domains (D, A, B and C) followed by a C-terminal region X, which anchors the protein to the cell wall of Staphylococcus aureus [14]. However, binding studies suggested that one molecule of intact SpA can only bind two molecules of IgG [I 9) leading to the hypothesis that SpA is functionally divalent in spite of its tetrameric structure [20].Recently a model for the formation of protein A-IgG soluble complexes has been proposed based on the evidence that IgG has two functional binding sites for SpA and that SPA has four functionally binding sites for IgG [21] nucleotide sequence analysis of the cloned SpA gene [22] reveals, however, a fifth region E, homologous to the four repetitive regions earlier identified by protein analysis. Sequence comparison between the repetitive IgG-binding regions show that region E has diverged more than the other four regions (D, A, B and C ) to a hypothetical consensus sequence [23]. ...