Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide

Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human alpha- to zeta-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained

Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-sequestering smooth endoplasmic reticulum in an invertebrate photoreceptor. I. Intracellular topography as revealed by OsFeCN staining and in situ Ca accumulation

Two ultrastructural approaches were used in photoreceptor cells of the leech, Hirudo medicinalis, to (a) investigate the intracellular topography of the smooth endoplasmic reticulum (SER) and (b) identify among the various subregions of the SER those which might function as Ca-sequestering sites. When the cells are prefixed with CaCl2-containing glutaraldehyde and postfixed with osmium tetroxide-ferricyanide (OsFeCN), only a part of the total SER is specifically stained. The stained SER cisternae include the submicrovillar cisternae (SMC), subsurface cisternae (SSC), the nuclear envelope, Golgi-associated SER, paracrystalline SER, and SER associated with glycogen areas. An extensive tubular SER cisternal system always remains unstained. When the cells are permeabilized by saponin and subsequently incubated with Ca2+, MgATP, and oxalate, the SMC (Walz, 1979, Eur. J. Cell Biol. 20:83-91), the SSC and the nuclear envelope contain electron-opaque Ca-oxalate precipitates indicating their ability to function as an effective Ca2+ sink. The results show that the very elaborate SER in this photoreceptor cell includes many functionally heterogeneous subregions. Of special physiological significance are those components (SMC and SSC) which are effective in Ca2+-buffering in the immediate vicinity of the plasma membrane.     

Transient Receptor Potential Channel 6 (TRPC6) Protects Podocytes during Complement-mediated Glomerular Disease

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis.     

Structural Basis for dsRNA Recognition,Filament Formation,and Antiviral Signal Activation by MDA5

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Highlights? MDA5 forms an open, C-shaped ring around the viral dsRNA stem ? The CTD of MDA5 has a different orientation and flexibility compared to RIG-I ? MDA5 forms filaments by stacking monomers head-to-tail with a 70° turn per monomer ? The 2CARD domain assembles into oligomers that activate interferon signaling via MAVS     

Probing functional perfection in substructures of ribonuclease T1: double combinatorial random mutagenesis involving Asn43, Asn44, and Glu46 in the guanine binding loop

Combinatorial random mutageneses involving either Asn43 with Asn44 (set 1) or Glu46 with an adjacent insertion (set 2) were undertaken to explore the functional perfection of the guanine recognition loop of ribonuclease T(1) (RNase T(1)). Four hundred unique recombinants were screened in each set for their ability to enhance enzyme catalysis of RNA cleavage. After a thorough selection procedure, only six variants were found that were either as active or more active than wild type which included substitutions of Asn43 by Gly, His, Leu, or Thr, an unplanned Tyr45Ser substitution and Glu46Pro with an adjacent Glu47 insertion. Asn43His-RNase T(1) has the same loop sequence as that for RNases Pb(1) and Fl(2). None of the most active mutants were single substitutions at Asn44 or double substitutions at Asn43 and Asn44. A total of 13 variants were purified, and these were subjected to kinetic analysis using RNA, GpC, and ApC as substrates. Modestly enhanced activities with GpC and RNA involved both k(cat) and K(M) effects. Mutants having low activity with GpC had proportionately even lower relative activity with RNA. Asn43Gly-RNase T(1) and all five of the purified mutants in set 2 exhibited similar values of k(cat)/K(M) for ApC which were the highest observed and about 10-fold that for wild type. The specificity ratio [(k(cat)/K(M))(GpC)/(k(cat)/K(M))(ApC)] varied over 30 000-fold including a 10-fold increase [Asn43His variant; mainly due to a low (k(cat)/K(M))(ApC)] and a 3000-fold decrease (Glu46Ser/(insert)Gly47 variant; mainly due to a low (k(cat)/K(M))(GpC)) as compared with wild type. It is interesting that k(cat) (GpC) for the Tyr45Ser variant was almost 4-fold greater than for wild type and that Pro46/(insert)Glu47 RNase T(1) is 70-fold more active than the permuted variant (insert)Pro47-RNase T(1) which has a conserved Glu46. In any event, the observation that only 6 out of 800 variants surveyed had wild-type activity supports the view that functional perfection of the guanine recognition loop of RNase T(1) has been achieved.     

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.     

InsP(3)-induced Ca(2+) release in permeabilized invertebrate photoreceptors: a link between phototransduction and Ca(2+) stores

Using the low-affinity fluorescent Ca(2+) indicators, Mag-Fura-2 and Mag-Fura Red, we studied light- and InsP(3)-induced Ca(2+) release in permeabilized microvillar photoreceptors of the medicinal leech, Hirudo medicinalis. Two major components of the phosphoinositide signaling pathway, phospholipase-C and the InsP(3) receptor, were characterized immunologically and appropriately localized in photoreceptors. Whereas phospholipase-C was abudantly expressed in photoreceptive microvilli, InsP(3) receptors were found mostly in submicrovillar endoplasmic reticulum (SER). Permeabilization of the peripheral plasma membrane with saponin allowed direct measurements of luminal free Ca(2+) concentration (Ca(L)) changes. Confocal Ca(2+) imaging using Mag-Fura Red demonstrated that Ins(1,4,5)P(3) mobilizes Ca(2+) from SER. As detected with Mag-Fura-2, a brief 50ms light flash activated rapid Ca(2+) depletion of SER, followed by an effective refilling within 1min of dark adaptation after the light flash. Sensitivity to Ins(1,4,5)P(3) of the Ca(2+) release from SER in leech photoreceptors was accompanied by irreversible uncoupling of phototransduction from Ca(2+) release. Depletion of Ca(2+) stores was induced by Ins(1,4,5)P(3)(EC(50)= 4.75 microM) and the hyper-potent agonist adenophostin A (EC(50)/40nM) while the stereoisomer L-myo Ins(1,4,5)P(3) was totally inactive. Ins(1,4,5)P(3)- or adenophostin A-induced Ca(2+) release was inhibited by 0.1-1mg/ml heparin. The Ca(2+) pump inhibitors, cyclopiazonic acid and thapsigargin, in the presence of Ins(1,4,5)P(3), completely depleted Ca(2+) stores in leech photoreceptors.