Growth retardation in mice lacking the proteasome activator PA28gamma

The proteasome activator PA28 binds to both ends of the central catalytic machine, known as the 20 S proteasome, in opposite orientations to form the enzymatically active proteasome. The PA28 family is composed of three members designated alpha, beta, and gamma; PA28alpha and PA28beta form the heteropolymer mainly located in the cytoplasm, whereas PA28gamma forms a homopolymer that predominantly occurs in the nucleus. Available evidence indicates that the heteropolymer of PA28alpha and PA28beta is involved in the processing of intracellular antigens, but the function of PA28gamma remains elusive. To investigate the role of PA28gamma in vivo, we generated mice deficient in the PA28gamma gene. The PA28gamma-deficient mice were born without appreciable abnormalities in all tissues examined, but their growth after birth was retarded compared with that of PA28gamma(+/-) or PA28gamma(+/+) mice. We also investigated the effects of the PA28gamma deficiency using cultured embryonic fibroblasts; cells lacking PA28gamma were larger and displayed a lower saturation density than their wild-type counterparts. Neither the expression of PA28alpha/beta nor the subcellular localization of PA28alpha was affected in PA28gamma(-/-) cells. These results indicate that PA28gamma functions as a regulator of cell proliferation and body growth in mice and suggest that neither PA28alpha nor PA28beta compensates for the PA28gamma deficiency.     

尿激酶原变体Glu151-Glu154-mscu-PA的构建及其动力学性质

利用寡核苷酸介导的定点突变方法 ,将重组人尿激酶原 ( recombinant single chain uroki-nase- type plasminogen activator,rscu- PA)中 1 51位赖氨酸 ( Lys1 51 )突变为谷氨酸 ( Glu1 51 ) ,1 54位精氨酸 ( Arg1 54)突变为谷氨酸 ( Glu1 54) ,得到尿激酶原变体基因 ( mscu- PA) .尿激酶原变体和未突变的重组尿激酶原均在 E.coli中获得表达 ,超声后所得包涵体经体外变复性并得到纯化 .结果表明 ,尿激酶原变体对纤溶酶 ( plasmin)的敏感性比未突变的重组尿激酶原低约 40 % ,转变纤溶酶原 ( Glu- plasminogen)为纤溶酶的活性基本相同 .两种产物经纤溶酶活化后 ,分别得到了双链尿激酶 ( rtcu- PA)和双链尿激酶变体 ( mtcu- PA) .它们对人工合成的发色底物 S2 4 4 4反应的动力学基本一致 ,对 Glu- plasminogen的催化反应的米氏动力学常数 Km 基本一致 ,但 mtcu- PA的 Kcat仅为rtcu- PA的 80 % .酪蛋白降解系统 ( caseinolytic system)实验表明 ,在纤维蛋白和纤溶酶原存在的情况下 ,尿激酶原变体较未突变尿激酶原能加快酪蛋白的降解 ,说明 mtcu- PA对纤维蛋白有一定的亲和性     

Preparation and characterization of two major isotypes of parvalbumins from skeletal muscle of bullfrog (Rana catesbeiana)

Two major isotypes of parvalbumins (PA1 and PA2) have been isolated from the skeletal muscle of bullfrog, Rana catesbeiana. The Mr values were estimated to be 10,100 (PA1) and 11,800 (PA2) by SDS/polyacrylamide gel electrophoresis, and the isoelectric points were determined to be 4.78 (PA1) and 4.97 (PA2) by polyacrylamide gel isoelectric focusing. The amino acid compositions and isoelectric points indicate that PA1 corresponds to Rana esculenta pI 4.50 and Rana temporaria pI 4.75 parvalbumins and PA2 to Rana esculenta pI 4.88 and Rana temporaria pI 4.97 parvalbumins, showing that PA1 is genetically a beta-parvalbumin and PA2 an alpha-parvalbumin. However, in terms of the amino acid compositions, PA1 and PA2 are distinctly different from the corresponding parvalbumins of Rana esculenta or Rana temporaria. The ultraviolet spectra of PA1 and PA2 are consistent with their amino acid compositions. An ultraviolet difference spectrum of the Ca2+-loaded form vs. metal-free form indicates that a Tyr and some Phe residues in PA1 are affected by a conformational change associated with the binding of Ca2+. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the Ca2+-loaded form of PA1 migrated twice as fast as the Mg2+-loaded form. Both PA1 and PA2 show increased mobility in the Ca2+-loaded forms, like troponin C but different from calmodulin.     

Regulation of phospholipase C-beta activity by phosphatidic acid: isoform dependence,role of protein kinase C,and G protein subunits

Phosphatidic acid (PA) stimulates phospholipase C-beta(1) (PLC-beta(1)) activity and promotes G protein stimulation of PLC-beta(1) activity. The isoform dependence for PA regulation of PLC-beta activity as well as the role of PA in modulating regulation of PLC-beta activity by protein kinase C (PKC) and G protein subunits was determined. As compared to PLC-beta(1), the phospholipase C-beta(3) (PLC-beta(3)) isoform was less sensitive to PA, requiring greater than 15 mol % PA for stimulation. PLC-beta(3) bound weakly to PA. PKC had little effect on PA stimulation of PLC-beta(3) activity. PKC, however, inhibited PA stimulation of PLC-beta(1) activity through a mechanism dependent on the mol % PA. Stimulation by 7.5 mol % PA was completely inhibited by PKC. Increasing the PA and Ca(2+) concentration attenuated PKC inhibition. The binding of PLC-beta(1) to PA containing phospholipid vesicles was also reduced by PKC, in a manner dependent on the mol % PA. PA increased the stimulation of PLC-beta(1) activity by G alpha q but had little effect on the stimulation by beta gamma subunits. These results demonstrate that PA stimulation of PLC-beta activity is tightly regulated, suggesting the existence of a distinct PA binding region in PLC-beta(1). PA may be an important component of a receptor mediated signaling mechanism that determines PLC-beta(1) activation.     

Increased palmitate intake: higher acylcarnitine concentrations without impaired progression of β-oxidation

Palmitic acid (PA) is associated with higher blood concentrations of medium-chain acylcarnitines (MCACs), and we hypothesized that PA may inhibit progression of FA β-oxidation. Using a cross-over design, 17 adults were fed high PA (HPA) and low PA/high oleic acid (HOA) diets, each for 3 weeks. The [1-¹³C]PA and [13-¹³C]PA tracers were administered with food in random order with each diet, and we assessed PA oxidation (PA OX) and serum AC concentration to determine whether a higher PA intake promoted incomplete PA OX. Dietary PA was completely oxidized during the HOA diet, but only about 40% was oxidized during the HPA diet. The [13-¹³C]PA/[1-¹³C]PA ratio of PA OX had an approximate value of 1.0 for either diet, but the ratio of the serum concentrations of MCACs to long-chain ACs (LCACs) was significantly higher during the HPA diet. Thus, direct measurement of PA OX did not confirm that the HPA diet caused incomplete PA OX, despite the modest, but statistically significant, increase in the ratio of MCACs to LCACs in blood.     

Reconstitution of hybrid proteasomes from purified PA700-20 S complexes and PA28alphabeta activator: ultrastructure and peptidase activities.

The activity of the proteasome, the major non-lysosomal proteinase in eukaryotes, is stimulated by two activator complexes, PA700 and PA28. PA700-20 S-PA700 proteasome complexes, generally designated as 26 S proteasomes, degrade proteins, whereas complexes of the type PA28-20 S-PA28 degrade only peptides. We report, for the first time, the in vitro reconstitution of previously identified hybrid proteasomes (PA700-20 S-PA28) from purified PA700-20 S proteasome complexes and PA28 activator. In electron micrographs, the hybrid appears as a corkscrew-shaped particle with a PA700 and a PA28 activator each bound to a terminal alpha-disk of the 20 S core proteasome. The multiple peptidase activities of hybrid proteasomes are not different from those of PA28-20 S-PA28 or PA700-20 S-PA700 complexes.     

Analogues of a potent oxytocin antagonist with truncated C-terminus or shorter amino acid side chain of the basic amino acid at position 8.

Twelve analogues were synthesized, their structure derived from modifications of [(S)Pmp1, D-Trp2, Pen6, Arg8]oxytocin, PA, in which (S)Pmp = beta,beta-(3-thiapentamethylene-beta-mercaptopropionic acid). PA is a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 8.86) and in the baboon. Truncated analogues of PA from the C-terminus were systematically prepared ending in either the free acid or the amide, i.e. PA1-9 acid, PA1-8 acid, PA1-7 acid, PA1-6 acid, PA1-8 amide, PA1-7 amide and PA1-6 amide. PA1-8 amide was roughly as potent as PA in the rat uterotonic assay in vitro, and the shorter amides were only somewhat weaker antagonists. All four acid analogues were weaker antagonists than PA but still maintained rather high antagonistic potency. These findings suggest that, if these truncated acids form as metabolites in vivo, they may contribute to the overall biological effect of PA and their contribution should be taken into account. Furthermore, using these analogues, the radioimmunoassay measurements of PA may be standardized, as they may cross react with PA antibodies and interfere with the determination. In addition, five analogues were made by substituting Arg8 of PA with Lys, Orn8, Dab8, Dap8 and Cit8. All of these analogues maintained high potency as OTAs in the uterotonic assay, although their activity was only about 1.5-3 times lower than PA. The most potent analogue in the uterotonic assay, [Dap8]PA, pA2 = 8.53, had weak pressor activity (pA2 = 6.90) and no antidiuretic effect. The pressor activity was lower for all tested acids, and for PA1-6 acid it was even below the detection limit. Additionally, PA1-9 acid, PA1-7 acid and PA1-6 acid showed no antidiuretic activity. Hence, the PA1-6 acid is a potent OTA with pA2 = 8.27 and no measurable effect in the pressor or antidiuretic tests and thus it is a pure oxytocin antagonist. This fact makes it an attractive candidate for further studies on inhibition of OT biological effects and on preterm labour.     

Hormonal stimulation alters the type of plasminogen activator produced by Sertoli cells

Primary cultures of immature rat Sertoli cells, maintained in serum-free medium, secrete two types of plasminogen activator (PA). When cultured under basal conditions, the preparations predominantly produce PA having a relative molecular weight (Mr) of 45,000 to 48,000. This PA activity is inactivated by antiserum against urokinase-type PA. When Sertoli cells are stimulated by follicle-stimulating hormone (FSH) or by dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), PA secretion is increased. The PA produced under these conditions has an Mr of 70,000, and is inactivated by antiserum against tissue-type PA but not by antiserum against urokinase-type PA. We conclude that, under basal conditions, Sertoli cells primarily secrete PA having the characteristics of urokinase-like PA (mu PA), and that Sertoli cells stimulated by FSH or by dbcAMP predominantly produce PA having the properties of tissue-type PA (tPA). Segments of adult rat seminiferous tubules, at defined stages of the cycle of the seminiferous epithelium, also produce and secrete two types of PA into the medium when maintained in organ culture. Segments at all stages examined release primarily mu PA in preparations cultured under basal conditions. In contrast, segments cultured in the presence of FSH synthesize larger amounts of PA, predominantly of the tPA type. An additional protease, which is independent of plasminogen, is secreted by tubule segments stimulated by FSH. The activity of this novel protease is not detectable in cultures maintained under basal conditions. We discuss the data in relation to the possible role of proteases in the restructuring of the seminiferous tubule during spermatogenesis.     

Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis

Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO(4)) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds (tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3, tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutants tds1, tds2, tds3, and tds5 (which do not produce PA) and tds6 (6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 and tt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation.     

Muscle parvalbumin isoforms of Clarias gariepinus, Heterobranchus longifilis and Chrysichthys auratus: isolation, characterization and expression during development

The white-muscle parvalbumin isoforms of Clarias gariepinus, Heterobranchus longifilis and Chrysichthys auratus were purified and their physicochemical parameters determined. The three catfish isoforms are distinct but those of C. gariepinus and H. longifilis are more similar. In the course of development, the successive appearance of larval and adult parvalbumins was observed. Larval isoforms (PA I, PA IIa, PA IIb) displayed a lower isoelectric point (pI) and molecular mass than adult ones (PA IIc, PA IIIa, PA IIIb, PA III, PA IV). The PA IIa isoform appeared as an omnipresent typical larval isoform. PA IIb appeared mostly larval, being insignificant in adult specimens; its physicochemical features were the same in the three catfish species. In Chrysichthys auratus , there were three PA II isoforms, one appearing as an adult isoform (PA IIc). The fact that the two types of parvalbumin isoforms appear at different times should reflect specific physiological needs (mobility, feeding) of different developmental stages.     

Both PA63 and PA83 are endocytosed within an anthrax protective antigen mixed heptamer: a putative mechanism to overcome a furin deficiency

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.     

利用大肠杆菌表达禽流感病毒聚合酶酸性蛋白

目的：通过在大肠杆菌中分段表达禽流感病毒聚合酶酸性蛋白（PA蛋白）,探索PA基因中可能影响表达的区域。方法：构建分段缺失的PA蛋白突变体,用IPTG在大肠杆菌RosettaGamiB（DE3）中诱导表达,比较各突变体的表达效率。结果：N端缺失长度在143～408个氨基酸残基之间的9个突变体在大肠杆菌中的表达水平较高;而突变体PA/K（Δ1-40aa）、PA/M（Δ1-56aa）、PA/N（Δ41-56aa）和PA/P（Δ57-75aa）的表达水平很低;全长PA蛋白和缺失N端20个氨基酸残基的突变体PA/L则检测不到表达。结论：PA基因的61～225bp和325～426bp可能是影响PA蛋白表达的2个重要区域,为下一步表达全长PA蛋白奠定了基础。     

Inhibition of the protease activity of influenza virus RNA polymerase PA subunit by viral matrix protein

Influenza virus PA is a subunit of RNA-dependent RNA polymerase. We demonstrated that PA has a unique chymotrypsin-like serine protease activity with Ser624 as an active site. To obtain further insight into the role of the protease activity of PA in viral proliferation, we examined the interaction between PA and matrix protein (M1). Both M1 purified from virion and hexa-histidine-tagged M1 expressed in Escherichia coli bound to PA. Hexa-histidine-tagged M1 pulled down PA. The interaction of PA with M1 was sensitive to ionic strength, suggesting that the interaction is formed by electrostatic force. Using Suc-Leu-Leu-Val-Tyr-MCA, a specific substrate for PA protease, M1 was demonstrated to inhibit the amidolytic activity of PA, whereas M1 did not inhibit that of chymotrypsin or trypsin at all. These results suggest that M1 binds to and inhibits the amidolytic activity of PA.     

Cations Mediate Interactions between the Nicotinic Acetylcholine Receptor and Anionic Lipids

Interactions between the nicotinic acetylcholine receptor (nAChR) and phosphatidic acid (PA) are bidirectional in that membranes containing PA are effective at stabilizing an agonist-responsive nAChR, whereas incorporation of the nAChR into the same membranes leads to a substantial increase in lipid lateral packing density. A previous study suggested that the ability of PA to adopt a dianionic ionization state is key. We monitored the ionization state of PA in both reconstituted and protein-free membranes. In model membranes composed of PA and 3:2 (mol/mol) phosphatidylcholine (PC)/PA, the monoanionic-to-dianionic transition of PA was detected with a pKa of 8.7 and 6.5, respectively. In the reconstituted 3:2 PC/PA membranes, however, PA was stabilized in a monoanionic state at pH values up to 10. Although dianionic PA does not play a role in nAChR function, we found that both the stabilization of monoanionic PA and the concentration of other cations at the bilayer surface can account for changes in bilayer physical properties that are observed upon incorporation of the nAChR into 3:2 PC/PA membranes. A nAChR-induced concentration of cations at the bilayer surface likely mediates interactions between the nAChR and the anionic lipids in its membrane environment.     

Anthrax toxin protective antigen: low-pH-induced hydrophobicity and channel formation in liposomes

To probe the role of the protective antigen (PA) component of anthrax toxin in toxin entry into animals cells, we examined the membrane channel-forming properties and hydrophobicity of intact and trypsin-cleaved forms of the protein at various pH values. At neutral pH neither form caused release of entrapped K+ from unilamellar lipid vesicles. At pH values below 6.0, however, K+ was rapidly released upon addition of either the nicked PA (PAN) or the 63 kDa tryptic fragment of PA (PA63), which has been implicated in the toxin entry process. Under the same conditions intact PA exhibited only weak channel-forming activity, and PA20, the complementary tryptic fragment, showed no such activity. Both PA and PA63 exhibited enhanced hydrophobicity at acidic pH values, but the enhancement was greater and the pH threshold higher with PA63. Our findings indicate that proteolytic removal of PA20 from intact PA enables the residual protein, PA63, to adopt a conformation at mildly acidic pH values that permits it to insert readily and form channels in membranes. Thus acidic conditions within endocytic vesicles may trigger membrane insertion of PA63, which in turn promotes translocation of ligated effector moieties, edema factor or lethal factor, across the vesicle membrane into the cytosol.     

Cellular phosphatidic acid sensor, α-synuclein N-terminal domain,detects endogenous phosphatidic acid in macrophagic phagosomes and neuronal growth cones

Phosphatidic acid (PA) is the simplest phospholipid and is involved in the regulation of various cellular events. Recently, we developed a new PA sensor, the N-terminal region of α-synuclein (α-Syn-N). However, whether α-Syn-N can sense physiologically produced, endogenous PA remains unclear. We first established an inactive PA sensor (α-Syn-N-KQ) as a negative control by replacing all eleven lysine residues with glutamine residues. Using confocal microscopy, we next verified that α-Syn-N, but not α-Syn-N-KQ, detected PA in macrophagic phagosomes in which PA is known to be enriched, further indicating that α-Syn-N can be used as a reliable PA sensor in cells. Finally, because PA generated during neuronal differentiation is critical for neurite outgrowth, we investigated the subcellular distribution of PA using α-Syn-N. We found that α-Syn-N, but not α-Syn-N-KQ, accumulated at the peripheral regions (close to the plasma membrane) of neuronal growth cones. Experiments using a phospholipase D (PLD) inhibitor strongly suggested that PA in the peripheral regions of the growth cone was primarily produced by PLD. Our findings provide a reliable sensor of endogenous PA and novel insights into the distribution of PA during neuronal differentiation.     

Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization

Immunofluorescence and other methods have been used to probe the self-assembly and internalization of the binary toxin, anthrax lethal toxin (LeTx), in primary murine macrophages. Proteolytic activation of protective antigen (PA; 83 kDa, the B moiety of the toxin) by furin was the rate-limiting step in internalization of LeTx and promoted clearance of PA from the cell surface. A furin-resistant form of PA remained at the cell surface for at least 90 min. Oligomerization of receptor-bound PA63, the 63 kDa active fragment of PA, was manifested by its conversion to a pronase-resistant state, characteristic of the heptameric prepore form in solution. That oligomerization of PA63 triggers toxin internalization is supported by the observation that PA20, the complementary 20 kDa fragment of PA, inhibited clearance of nicked PA. The PA63 prepore, with or without lethal factor (LF), cleared slowly from the cell surface. These studies show that proteolytic cleavage of PA, in addition to permitting oligomerization and LF binding, also promotes internalization of the protein. The relatively long period of activation and internalization of PA at the cell surface may reflect adaptation of this binary toxin that maximizes self-assembly.