Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apolipoproteins share a common structural feature, their interaction with phospholipids. It is believed that amphipathic helical sequences enable apolipoproteins to bind to lipid bilayer and to form discoidal particles of defined dimensions. While the knowledge of the apo A-I sequence and secondary structure has been used to make predictions about its mode of association with lipids, the available experimental data necessary to propose a precise model of these discoidal structures are still limited. An important step in our understanding of these structures would be to identify the apolipoprotein lipid-associated domains. Proteolysis of apo A-I-DMPC reconstituted HDL (rHDL) and free apo A-I is used here to identify lipid-protected domains of apo A-I. Free cleaved peptides were separated from rHDL associated peptides by density gradient centrifugation. The lipid-associated peptides were further analyzed by SDS-PAGE and transferred by Western blot to a ProBlott membrane for sequencing. Cleavage occurred at residue 43 with proteinase K, 46 with trypsin and residue 47 or 48 with pronase. A large domain from about residue 45 to the C-terminal remains highly protected against hydrolysis eventhough it contains several bonds susceptible to proteolytic cleavage. No protected fragments were detected by SDS-PAGE after enzymatic cleavage of free apo A-I in identical experimental conditions.
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PMID:Enzymatic hydrolysis of reconstituted dimyristoylphosphatidylcholine-apo A-I complexes. 837 88

Estrogen-mediated accumulation of the avian apolipoprotein (apo) II mRNA is in part due to its stabilization. To identify the biochemical activity responsible for this effect, radiolabeled, capped, and polyadenylated apoII mRNA was incubated in vitro in liver cytosolic extracts from roosters who received either estrogen (estrogen-treated extract) or the vehicle (control extract) parenterally. The mRNA was very stable in estrogen-treated extract but was rapidly degraded in control extract. The RNA was degraded predominantly by endonuclease rather than exonuclease activity. The addition of the estrogen-treated extract to the control extract prevented the degradation of the mRNA in trans. This biochemical activity was heat labile and was also destroyed by proteinase K but not by micrococcal nuclease, indicating that estrogen treatment resulted in the expression of a protein in the liver that stabilized the apoII mRNA by inhibiting its nucleolytic degradation. This mRNA stabilization factor was labile around 60 degrees C, whereas the RNase remained stable up to 80 degrees C. Studies on mRNA protein interaction showed that both control and estrogen-treated extracts contain mRNA-binding (mRNP) proteins that bind apoII mRNA. An increased binding to apoII mRNA by a subset of these proteins was observed with estrogen-treated extract as compared with the control extract. This activity, although it afforded complete protection from nucleolytic degradation to apoII and apo A1 mRNAs, appeared to provide less protection to mRNAs encoding chicken serum albumin and vitellogenin, suggesting differential stabilization of mRNAs. These studies indicate that a cytosolic mRNA-stabilization factor, providing apoII mRNA complete protection from nucleolytic degradation, is expressed in the avian liver upon estrogen treatment. This appears to be the first time that a biochemical activity responsible for hormone-mediated stabilization of mRNAs and estrogen induction of mRNA binding by specific mRNPs have been identified and partially characterized in vitro.
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PMID:In vitro characterization of an estrogen-regulated mRNA stabilizing activity in the avian liver. 877 38

Exchangeable serum apolipoproteins and amphipathic alpha-helical peptides are effective inhibitors of sterol (free and esterified cholesterol) uptake at the small-intestinal brush border membrane. The minimal structural requirement of an inhibitor is an amphipathic alpha-helix of 18 amino acids. The inhibition is competitive, indicating that the inhibitor binds to scavenger receptor class B type I (SR-BI) present in the brush border membrane and responsible for sterol uptake. Binding of apolipoprotein A-I to SR-BI of rabbit brush border membrane is cooperative, characterized by a dissociation constant K(d) = 0.45 microM and a Hill coefficient of n = 2.8. The cooperativity of the interaction is due to binding of the inhibitor molecule to a dimeric or oligomeric form of SR-BI held together by disulfide bridges. Consistent with the competitive nature of the inhibition, the K(d) value agrees within experimental error with the IC(50) value of inhibition and with the inhibition constant K(I). After proteinase K treatment of brush border membrane vesicles, the affinity of the interaction of apolipoprotein A-I expressed as K(d) is reduced by a factor of 20, and the cooperativity is lost. The interaction of proteinase K-treated brush border membrane vesicles with apolipoprotein A-I is nonspecific partitioning of the apolipoprotein into the lipid bilayer of brush border membrane vesicles.
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PMID:Intestinal sterol absorption mediated by scavenger receptors is competitively inhibited by amphipathic peptides and proteins. 1102 42

There is increasing epidemiologic evidence implying a role for chronic infection in atherosclerosis and that microbial TLR agonists may contribute to this disease. Mycoplasma arthritidis is an agent of acute and chronic inflammatory disease in rodents, and has been used extensively as a model for defining the mechanisms involved in arthritis and other inflammatory diseases. We have purified a 28-kDa, apolipoprotein A-1 (apoA-1)-like TLR2-dependent macrophage-activating moiety from a culture of a virulent strain of M. arthritidis. ApoA-1 similarly isolated from uninoculated mycoplasma medium was without bioactivity. The activity of the mycoplasma-derived molecule was resistant to heat and to digestion with proteinase K, but was susceptible to alkaline hydrolysis and H(2)O(2) oxidation. Infrared profiles of normal apoA-1 and that derived from mycoplasma were distinct. Unlike the activity of other mycoplasmal TLR2 agonists such as macrophage-activating lipopeptide-2, activity of the M. arthritidis-derived 28-kDa component was dependent upon CD14, a coreceptor for LPS. Finally, we showed that bioactive lipopeptides prepared from M. arthritidis grown in serum-free medium and also from a 41-kDa known bioactive lipoprotein of M. arthritidis, avidly bound to purified apoA-1 that separated out by SDS-PAGE, induced TNF-alpha and IL-12p40 both in vitro and in vivo. ApoA-1 is a key functional component of the high-density lipoprotein cholesterol complex by scavenging and removing unwanted lipids. Our finding that this molecule can acquire macrophage-activating properties from microbial TLR2-dependent agonists suggests a novel mechanism whereby some microbial agents might reverse the protective role of apoA-1, thus contributing to the genesis of atherosclerosis.
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PMID:A microbial TLR2 agonist imparts macrophage-activating ability to apolipoprotein A-1. 1698 24

Plasma apolipoproteins show alpha-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19-37 and 57-74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II(56-76). Synthetic apoC-II(56-76) readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60-70. We postulate that the ability of apoC-II(60-70) to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo.
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PMID:A structural core within apolipoprotein C-II amyloid fibrils identified using hydrogen exchange and proteolysis. 1721 59

Mycoplasma arthritidis is a naturally occurring murine pathogen, and the disease model has been used extensively to understand inflammatory mechanisms. Recently, Triton X-114 extracts of a virulent strain of M. arthritidis were found to be more potent in activating macrophages than were those from an avirulent strain, suggesting a role in disease. Here, octyl glucoside extraction of cells was used to identify four distinct bioactive moieties, with molecular masses of approximately 41, 37, 34, and 17 kDa. Their bioactivities were resistant to proteinase K but were destroyed by alkaline hydrolysis and oxidation. As for MALP-2, all were dependent upon Toll-like receptor 2, but unlike MALP-2, they were also dependent upon CD14. The M. arthritidis lipoproteins exhibited infrared absorbances at 2,900 cm(-1) and 1,662 cm(-1), similar to those seen in Pam(3)-Cys-Ser-(Lys)(4). Edman degradation failed to reveal N-terminal sequences, suggesting that they were blocked and therefore might be triacylated. However, mass spectrometry of fragments revealed that the 41-kDa moiety, which binds to serum apolipoprotein A-1, had similarity with the recently described MlpD lipoprotein of M. arthritidis.
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PMID:Inflammatory lipoproteins purified from a toxigenic and arthritogenic strain of Mycoplasma arthritidis are dependent on Toll-like receptor 2 and CD14. 1728 6