Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss

Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1beta is poorly understood. In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.     

Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex

Phosphatidylinositol 4-kinasebeta (PI4Kbeta) plays an essential role in maintaining the structural integrity of the Golgi complex. In a search for PI4Kbeta-interacting proteins, we found that PI4Kbeta specifically interacts with the GTP-bound form of the small GTPase rab11. The PI4Kbeta-rab11 interaction is of functional significance because inhibition of rab11 binding to PI4Kbeta abolished the localization of rab11 to the Golgi complex and significantly inhibited transport of vesicular stomatitis virus G protein from the Golgi complex to the plasma membrane. We propose that a novel function of PI4Kbeta is to act as a docking protein for rab11 in the Golgi complex, which is important for biosynthetic membrane transport from the Golgi complex to the plasma membrane.     

High level of male-biased Scandinavian admixture in Greenlandic Inuit shown by Y-chromosomal analysis

We have used binary markers and microsatellites on the Y chromosome to analyse diversity in a sample of Greenlandic Inuit males. This sample contains Y chromosomes typical of those found in European populations. Because the Y chromosome has a unique and robust phylogeny of a time depth that precedes the split between European and Native American populations, it is possible to assign chromosomes in an admixed population to either continental source. On this basis, 58+/-6% of these Y chromosomes have been assigned to a European origin. The high proportion of European Y chromosomes contrasts with a complete absence of European mitochondrial DNA and indicates strongly male-biased European admixture into Inuit. Comparison of the European component of Inuit Y chromosomes with European population data suggests that they have their origins in Scandinavia. There are two potential source populations: Norse settlers from Iceland, who may have been assimilated 500 years ago, and the Danish-Norwegian colonists of the eighteenth century. Insufficient differentiation between modern Icelandic and Danish Y chromosomes means that a choice between these cannot be made on the basis of diversity analysis. However, the extreme sex bias in the admixture makes the later event more likely as the source.     

Effects of truncated angiotensins in humans after double blockade of the renin system

Angiotensins different from ANG II exhibit biological activities, possibly mediated via receptors other than ANG II receptors. We studied the effects of 3-h infusions of ANG III, ANG-(1-7), and ANG IV in doses equimolar to physiological amounts of ANG II (3 pmol. kg-1. min-1), in six men on low-sodium diet (30 mmol/day). The subjects were acutely pretreated with canrenoate and captopril to inhibit aldosterone actions and ANG II synthesis, respectively. ANG II infusion increased plasma angiotensin immunoreactivity to 53 +/- 6 pg/ml (+490%), plasma aldosterone to 342 +/- 38 pg/ml (+109%), and blood pressure by 27%. Glomerular filtration rate decreased by 16%. Concomitantly, clearance of endogenous lithium fell by 66%, and fractional proximal reabsorption of sodium increased from 77 to 92%; absolute proximal reabsorption rate of sodium remained constant. ANG II decreased sodium excretion by 70%, potassium excretion by 50%, and urine flow by 80%, whereas urine osmolality increased. ANG III also increased plasma aldosterone markedly (+45%), however, without measurable changes in angiotensin immunoreactivity, glomerular filtration rate, or renal excretion rates. During vehicle infusion, plasma renin activity decreased markedly ( approximately 700 to approximately 200 mIU/l); only ANG II enhanced this decrease. ANG-(1-7) and ANG IV did not change any of the measured variables persistently. It is concluded that 1) ANG III and ANG IV are cleared much faster from plasma than ANG II, 2) ANG II causes hypofiltration, urinary concentration, and sodium and potassium retention at constant plasma concentrations of vasopressin and atrial natriuretic peptide, and 3) a very small increase in the concentration of ANG III, undetectable by usual techniques, may increase aldosterone secretion substantially.     

Reactive oxygen species are important mediators of taurine release from skeletal muscle cells

The present study illustrates elements ofthe signal cascades involved in the activation of taurine effluxpathways in myotubes derived from skeletal muscle cells. Exposingprimary skeletal muscle cells, loaded with ¹⁴C-taurine, to1) hypotonic media, 2) the phospholipaseA₂ (PLA₂) activator melittin, 3)anoxia, or 4) lysophosphatidyl choline (LPC) causes anincrease in ¹⁴C-taurine release and a concomitantproduction of reactive oxygen species (ROS). The antioxidants butulatedhydroxy toluene and vitamin E inhibit the taurine efflux after cellswelling, anoxia, and addition of LPC. The muscle cells possess twoseparate taurine efflux pathways, i.e., a swelling- andmelittin-induced pathway that requires 5-lipoxygenase activity foractivation and a LPC-induced pathway. The two pathways aredistinguished by their opposing sensitivity toward the anion channelblocker DIDS and cholesterol. These data provide evidence forPLA₂ products and ROS as key mediators of the signalcascade leading to taurine efflux in muscle.

     

A low-barrier hydrogen bond between histidine of secreted phospholipase A2 and a transition state analog inhibitor

This work describes in-depth NMR characterization of a unique low-barrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A(2) (sPLA(2), from bee venom and bovine pancreas) and a transition-state analog inhibitor HK32. A downfield proton NMR resonance, at 17-18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48. These results led to a hypothesis that the downfield resonance represents the proton (H(epsilon 2) of H48) involved in the H-bonding between D99 and H48, in analogy with serine proteases. However, this was shown not to be the case by use of the bovine enzyme labeled with specific [15N(epsilon 2)]His. Instead, the downfield resonance arises from H(delta1) of H48, which forms a hydrogen bond with a non-bridging phosphonate oxygen of the inhibitor. Further studies showed that this proton displays a fractionation factor of 0.62(+/-0.06), and an exchange rate protection factor of >100 at 285 K and >40 at 298 K, which are characteristic of a LBHB. The pK(a) of the imidazole ring of H48 was shown to be shifted from 5.7 for the free enzyme to an apparent value of 9.0 in the presence of the inhibitor. These properties are very similar to those of the Asp em leader His LBHBs in serine proteases. Possible structural bases and functional consequences for the different locations of the LBHB between these two types of enzymes are discussed. The results also underscore the importance of using specific isotope labeling, rather than extrapolation of NMR results from other enzyme systems, to assign the downfield proton resonance to a specific hydrogen bond. Although our studies did not permit the strength of the LBHB to be accurately measured, the data do not provide support for an unusually strong hydrogen bond strength (i.e. >10 kcal/mol).     

TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.     

Processing of seminal plasma hCAP-18 to ALL-38 by gastricsin: a novel mechanism of generating antimicrobial peptides in vagina

The human cathelicidin, hCAP-18, is expressed both in neutrophils and in epithelial cells. hCAP-18 is processed to the antimicrobial peptide LL-37 by proteinase 3 in neutrophils. hCAP-18 is highly expressed in the epididymis with a subsequent high concentration in seminal plasma where the protein is present in its unprocessed and antimicrobially inactive form. We report here that hCAP-18 in seminal plasma is processed to generate a 38-amino acid antimicrobial peptide ALL-38 by the prostate-derived protease gastricsin when incubated at a pH corresponding to the vaginal pH. In accordance with this, seminal plasma derived hCAP-18 was found in its processed form in the vagina following sexual intercourse. The antimicrobial activity of ALL-38 against a variety of microorganisms tested is equal to that of LL-37. This enzymatic activation of a proantimicrobial substance in seminal plasma following exposure to the vaginal milieu represents a novel mechanism to prevent infection following sexual intercourse.     

Misfolding, degradation, and aggregation of variant proteins. The molecular pathogenesis of short chain acyl-CoA dehydrogenase (SCAD) deficiency

Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding, and preliminary experiments suggested that the variant protein displayed prolonged association with chaperonins and delayed formation of active enzyme. Accordingly, the molecular pathogenesis of SCAD deficiency may rely on intramitochondrial protein quality control mechanisms, including degradation and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate with mitochondrial hsp60 chaperonins; however, the variant SCAD proteins remained associated with hsp60 for prolonged periods of time. Biogenesis experiments at two temperatures revealed that some of the variant proteins (R22W, G68C, W153R, and R359C) caused severe misfolding, whereas others (R147W, G185S, and Q341H) exhibited a less severe temperature-sensitive folding defect. Based on the magnitude of in vitro defects, these SCAD proteins are characterized as folding-defective variants and mild folding variants, respectively. Pulse-chase experiments demonstrated that the variant SCAD proteins either triggered proteolytic degradation by mitochondrial proteases or, especially at elevated temperature, aggregation of non-native conformers. The latter finding may indicate that accumulation of aggregated SCAD proteins may play a role in the pathogenesis of SCAD deficiency.