Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several studies have demonstrated that lipid-free apolipoproteins can promote cholesterol and phospholipid efflux from cells; however, the mechanisms and the role of cell-mediated pathways involved remain incompletely elucidated. We have recently demonstrated that brefeldin A or monensin, agents that disrupt Golgi apparatus structure and function, inhibit intracellular cholesterol efflux from cells to high density lipoproteins. In the present study we examined the effects of those agents on cell cholesterol and phospholipid efflux to purified apolipoprotein A-I (apoA-I) and apolipoprotein-depleted acceptors from cholesterol-loaded fibroblasts. Brefeldin A or monensin treatment of cells during incubation with apoA-I inhibited efflux of cellular cholesterol by greater than 80% compared with control cells, measured by changes in cellular cholesterol radioactivity, mass, and the substrate pool of cholesterol available for esterification by acyl coenzyme A:cholesterol acyltransferase. Inhibition of cholesterol efflux by these agents could not be overcome by increasing the apoA-I concentration and persisted during incubations up to 24 h. Similarly, brefeldin A and monensin inhibited up to 80% of apoA-I-mediated efflux of labeled phospholipids from cholesterol-loaded cells relative to controls. In contrast, lipid efflux mediated by apolipoprotein-depleted acceptors (trypsin-modified HDL and sonicated phospholipid vesicles) was not sensitive to these drugs. On the basis the known effects of brefeldin A and monensin on Golgi apparatus structure and function, these results are consistent with the notion that efflux of cell lipids by apolipoprotein-dependent mechanisms, but not by apolipoprotein-independent mechanisms, require active cellular processes involving an intact and functional Golgi apparatus.
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PMID:Apolipoprotein-mediated cellular cholesterol and phospholipid efflux depend on a functional Golgi apparatus. 901 4

High-density lipoprotein (HDL) components remove cholesterol from cells by two independent mechanisms. Whereas HDL phospholipids pick up cholesterol that desorbs from the plasma membranes, HDL apolipoproteins appear to interact with cell-surface binding sites that target for removal pools of cellular cholesterol that feed into the cholesteryl ester cycle. Here we show that mild trypsin treatment of HDL almost completely abolishes this apolipoprotein-mediated cholesterol removal process. When HDL was treated with trypsin for various periods of time and then incubated with cholesterol-loaded fibroblasts, treatment for only 5 min reduced the ability of HDL to remove excess cholesterol from cellular pools that were accessible to esterification by the enzyme acyl CoA:cholesterol acyltransferase. This mild treatment digested less than 20% of HDL apolipoproteins and did not alter the lipid composition, size distribution, or electrophoretic mobility of the particles. Trypsin treatment of HDL for up to 1 h caused no further reduction in its ability to remove cellular cholesterol despite a greater than 2-fold increase in apolipoprotein digestion. Trypsin treatment of HDL also reduced its ability to deplete the cholesteryl ester content of sterol-laden macrophages. Promotion of cholesterol efflux from the plasma membrane by HDL phospholipids was unaffected by even extensive proteolysis. In parallel to the loss of cholesterol transport-stimulating activity, trypsin treatment of HDL for only 5 min nearly abolished its interaction with high-affinity binding sites on cholesterol-loaded fibroblasts. Reconstitution of trypsin-modified HDL with isolated apo A-I or apo A-II restored the cholesterol transport-stimulating activity of the particles. Thus a minor trypsin-labile fraction of HDL apolipoproteins is almost exclusively responsible for the apolipoprotein-dependent component of cholesterol efflux mediated by HDL particles.
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PMID:Limited proteolysis of high density lipoprotein abolishes its interaction with cell-surface binding sites that promote cholesterol efflux. 921 13

It is becoming increasingly accepted that removal of cellular cholesterol occurs by at least two pathways, one involving the well-described aqueous diffusion mechanism and another promoted by lipid-free apolipoproteins. We compared the contribution of apolipoprotein-dependent and -independent pathways, taking into consideration the influence of cellular metabolism, on cholesterol efflux promoted by different extracellular acceptor types. The acceptors used were assumed to participate in only passive efflux by lipid-dependent mechanisms (phospholipid vesicles and trypsin-modified high density lipoproteins) or to stimulate efflux by apolipoprotein-dependent pathways (purified apolipoprotein A-I and high density lipoproteins). Apolipoprotein-mediated cholesterol efflux was only apparent in growth-arrested or cholesterol-enriched cells and required metabolic energy. In contrast, cholesterol efflux by apolipoprotein-depleted acceptors did not depend on cell growth state, cholesterol enrichment, or metabolic energy. Apolipoprotein-mediated efflux was not observed at temperatures below 22 degrees C, while apolipoprotein-independent efflux was only reduced by 50% at 4 degrees C compared with incubations at 37 degrees C. Additionally, apolipoproteins promoted a more rapid and larger decrease in intracellular cholesteryl esters when measured by changes in cholesteryl ester radioactivity, mass, or the pool of cholesterol available for esterification by acyl coenzyme A:cholesterol acyltransferase. Efflux of excess cellular cholesterol by an apolipoprotein-dependent pathway appears to involve specific cellular events consistent with the properties of an active transport pathway and distinguishable from cholesterol efflux by apolipoprotein-depleted acceptors through passive mechanisms.
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PMID:Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion. 932 90

Protein kinase C (PKC) seems to play an important role in many of HDL effects on cells, including removal of excess cholesterol. HDL removes cholesterol by at least two mechanisms. One mechanism involves desorption/diffusion of cholesterol from the plasma membrane onto the acceptor particle, whereas the second is mediated by apolipoproteins and may involve intracellular translocation of cholesterol to the plasma membrane for subsequent efflux. In this report, we examined the possibility that mitogen-activated protein (MAP) kinase is one of the downstream events from HDL activation of PKC. Using a gel kinase assay with myelin basic protein incorporated into the gel, HDL (50 micrograms protein/mL) stimulated multiple kinases of 42, 50, 52, 58, and 60 kDa. The 42-kDa protein kinase, corresponding to the unresolved MAP kinases ERK1 and ERK2 based on immunoblotting, was activated over 2-fold by HDL. HDL activated all identified kinases in a concentration- and time-dependent manner, which became maximal within 5 to 10 minutes and remained activated for at least 60 minutes. HDL activation of MAP kinase seems to be partially mediated by PKC, because down-regulation of PKC and known PKC inhibitors inhibited the HDL effect by 40 to 50%. Free apolipoproteins A-I (10 micrograms/mL) and A-II (10 micrograms/mL) had no significant effect on MAP kinase activation. Moreover, modifying HDL with trypsin or tetranitromethane, which abolishes apolipoprotein-mediated cholesterol efflux, had no effect on HDL activation of MAP kinase. These results suggest that HDL activates MAP kinase via multiple signal transduction pathways that are likely involved in an HDL effect unrelated to apolipoprotein-mediated cholesterol translocation and efflux.
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PMID:High density lipoproteins stimulate mitogen-activated protein kinases in human skin fibroblasts. 932 61

Interaction of HDL with cells activates protein kinase C (PKC), a process that may be important in stimulating efflux of excess cellular cholesterol. Here we report that HDL treatment of cholesterol-loaded fibroblasts increases 32P labeling of three acidic phosphoproteins. These phosphoproteins, called pp80, pp27, and pp18 based on apparent M(r) in kD, were also phosphorylated by acute treatment of cells with phorbol myristate acetate, suggesting that they are regulated in response to PKC activation. The HDL-stimulated phosphorylation of pp80 and pp18 was significant after only 30 seconds and was sustained for at least 30 and 120 minutes, respectively, while increased phosphorylation of pp27 was transient, reaching a maximum at 10 minutes. Both pp27 and pp18 were phosphorylated on serine/threonine residues, whereas pp80 was phosphorylated on serine/threonine and tyrosine residues. Immunoprecipitation studies suggested that pp80 is the myristoylated alanine-rich C kinase substrate protein, but the identities of pp27 and pp18 are unknown. HDL and trypsin-digested HDL stimulated phosphorylation of pp80 and pp27, while purified apoA-I, apoA-II, or apoE had no stimulatory effects, indicating that the active component in HDL was trypsin resistant and unlikely to be an apolipoprotein. Conversely, HDL, apoA-I, apoA-II, and apoE all stimulated pp18 phosphorylation, while trypsin-digested HDL had less effect, consistent with pp18's being responsive to HDL apolipoproteins. Treatment of cholesterol-depleted cells with apoA-I also stimulated phosphorylation of pp18, but only transiently. These results suggest that HDL interaction with cells activates diverse PKC-mediated pathways that target different phosphoproteins. Of these three phosphoproteins, only pp18 has a phosphorylation response consistent with its being involved in apolipoprotein-mediated lipid transport.
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PMID:Phosphoproteins regulated by the interaction of high-density lipoprotein with human skin fibroblasts. 940 45

A 51-yr-old woman without clinical evidence of Tangier disease, but with an extremely low high density lipoprotein (HDL) cholesterol level, was studied. No defect in the major structural protein of HDL, apolipoprotein AI (apo AI), was detected. A preponderance of small HDL particles in the patient's plasma suggested defective uptake of cellular cholesterol. Efflux of [3H]cholesterol from patient fibroblasts to normal apo AI was decreased 50%. Cholesterol efflux to HDL was also decreased, but efflux to trypsin-modified HDL was not. The patient's cells partitioned more exogenously provided [3H]cholesterol into free cholesterol and synthesized greater amounts of phosphatidylcholine than did normal or Tangier fibroblasts. Her fibroblasts did not differ from normal fibroblasts in sterol synthesis rate, cellular cholesterol and cholesterol ester content, or incorporation of oleate into cholesterol ester. The data indicate the presence of a defect in apolipoprotein-dependent cellular cholesterol efflux that differs from that seen in Tangier disease. These findings are the first evidence that other low HDL cholesterol syndromes, besides Tangier disease, may also be associated with cholesterol efflux abnormalities. The identification of mutant genes responsible for apolipoprotein-mediated efflux abnormalities should provide valuable insights into cellular mechanisms involved in the reverse cholesterol transport pathway.
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PMID:Decreased cholesterol efflux from fibroblasts of a patient without Tangier disease, but with markedly reduced high density lipoprotein cholesterol levels. 950 37

Studies in different liver-derived cells in culture indicate that apolipoprotein (apo) B-100 production is regulated largely by intracellular degradation and the ubiquitin-proteasome pathway is a major mechanism for the degradation. The proteasomal degradation of apoB-100 was postulated to be an intrinsic property of the protein that occurs even in the presence of optimal amounts of lipids supplied to the cell. We examined apoB-100 and apoB-48 biogenesis in CaCo2, a human colon carcinoma cell line. To our surprise, apoB-100 and apoB-48 were quantitatively secreted by CaCo2 cells; essentially none of the newly synthesized apoB was degraded before secretion in a 2-h period whether the cells were cultured on filter or on plastic. Furthermore, although ubiquitin immunoreactivity was readily detected in the intracellular apoB isolated from HepG2 cells, little or no ubiquitin was detectable in the intracellular apoB from CaCo2 cells. The amounts of free ubiquitin and total and non-apoB ubiquitinated proteins were comparable in HepG2 and CaCo2 cells, indicating that CaCo2 cells have the necessary machinery for tagging ubiquitin chains onto cellular proteins for proteasomal degradation. Incubation in lipoprotein-deficient serum did not induce apoB degradation, but the addition of a microsomal triglyceride transfer protein inhibitor led to apoB degradation in CaCo2 cells. Finally, similar proportions of apoB polypeptide in isolated microsomes from CaCo2 and HepG2 cells were accessible to exogenously added trypsin, indicating that the mere exposure of apoB nascent chains to the cytosolic compartment is insufficient to cause the proteasomal degradation. Therefore, the intracellular degradation of apoB is not an intrinsic property of the protein, and the phenomenon is neither universal nor inevitable. The unconditional use of apoB as a paradigm for intracellular protein degradation is not warranted.
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PMID:Apolipoprotein B, a paradigm for proteins regulated by intracellular degradation, does not undergo intracellular degradation in CaCo2 cells. 1066 May 49

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is present in plasma as an apolipoprotein and as a cell-associated lipase. GPI-PLD mRNA levels are regulated, but it is unclear if posttranslational mechanisms also regulate GPI-PLD function. We examined the effect of protein kinase A phosphorylation on human serum GPI-PLD activity, trypsin activation, and apolipoprotein AI binding. Protein kinase A phosphorylation did not activate GPI-PLD activity in vitro, nor did phosphorylated GPI-PLD cleave a GPI-anchored protein from intact porcine erythrocytes. Trypsin cleaves the C-terminal beta propeller of purified human serum GPI-PLD to generate three immunodetectable fragments (75, 28, and 18 kDa) in association with a 12-fold increase in enzyme activity. After phosphorylation, the amounts of 28- and 18-kDa fragments were markedly decreased with trypsin treatment, and activity was only increased five-fold. Phosphorylation also inhibits binding of GPI-PLD to apolipoprotein AI. These data are the first demonstrating that phosphorylation may regulate GPI-PLD interaction with other proteins.
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PMID:Phosphorylation decreases trypsin activation and apolipoprotein al binding to glycosylphosphatidylinositol-specific phospholipase D. 1198 19

A unique property of the extracellular matrix of J774 and THP-1 cells has been identified, which contributes to the ability of these cells to promote cholesterol efflux. We demonstrate high level apolipoprotein (apo) A-I binding to macrophage cells (THP-1 and J774) and to their extracellular matrix (ECM). However, high level apoA-I binding is not observed on fibroblasts, HepG2 cells, or U937 cells (a macrophage cell line that does not efflux cholesterol to apoA-I or bind apoA-I on their respective ECM). Binding to the ECM of THP-1 or J774 macrophages depends on the presence of apoA-I C-terminal helices and is markedly reduced with a mutant lacking residues 187-243 (apoA-I Delta(187-243)), suggesting that the hydrophobic C terminus forms a hydrophobic interaction with the ECM. ApoA-I binding is lost upon trypsin treatment or with Triton X-100, a preparation method that de-lipidates the ECM. However, binding is recovered with re-lipidation, and is preserved with ECM prepared using cytochalasin B, which conserves the endogenous phospholipid levels of the ECM. We also demonstrate that specific cholesterol efflux to apoA-I is much reduced in cells released from their native ECM, but fully restored when ECM-depleted cells are added back to ECM in the presence of apoA-I. The apoA-I-mediated efflux is deficient in plated or suspension U937 macrophages, but is restored to high levels when the suspension U937 cells are reconstituted with the ECM of J774 cells. The ECM-dependent activity was much reduced in the presence of glyburide, indicating participation of ABCA1 (ATP-binding cassette transporter 1) in the efflux mechanism. These studies establish a novel binding site for apoA-I on the macrophage ECM that may function together with ABCA1 in promoting cholesterol efflux.
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PMID:Trypsin-sensitive and lipid-containing sites of the macrophage extracellular matrix bind apolipoprotein A-I and participate in ABCA1-dependent cholesterol efflux. 1205 Jan 68

We show that murine macrophages that have ingested cell membranes as a source of cholesterol exhibit a marked increase in acyl-CoA:cholesterol acyl transferase (ACAT) activity. Exposure of these macrophages to acute-phase high-density lipoprotein (HDL) results in a marked reduction of ACAT and enhancement of cholesteryl ester hydrolase (CEH) activities, phenomena not seen with native HDL. These complementary but opposite effects of acute-phase HDL on the two enzyme systems that regulate the balance between esterified (storage) cholesterol and unesterified (transportable) cholesterol are shown to reside with serum amyloid A (SAA) 2.1, an acute-phase apolipoprotein of HDL whose plasma concentration increases 500- to 1,000-fold within 24 h of acute tissue injury. Mild trypsin treatment of acute-phase HDL almost completely abolishes the apolipoprotein-mediated effects on the cholesteryl ester cycle in cholesterol-laden macrophages. The physiological effect of SAA2.1 on macrophage cholesterol is to shift it into a transportable state enhancing its rate of export, which we confirm in tissue culture and in vivo. The export process is shown to be coupled to the ATP binding cassette transport system. Our findings integrate previous isolated observations about SAA into the sphere of cholesterol transport, establish a function for a major acute-phase protein, and offer a novel approach to mobilizing macrophage cholesterol at sites of atherogenesis.
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PMID:Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1. 1223 72


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