Enzymatic reduction of disulfide bonds in lysosomes: characterization of a gamma-interferon-inducible lysosomal thiol reductase (GILT)

Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal/lysosomal system by the mannose 6-phosphate receptor, N- and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-gamma in other cell types, suggesting a potentially important role in antigen processing.     

Egg maturation and ecdysiotropic activity in extracts of mosquito (Aedes aegypti) heads

Blood-fed, decapitated female Aedes aegypti mosquitoes matured eggs when injected with an extract of heads, but non-blood-fed females did not. The response to head extract was optimal when the head was allowed to remain for 2 hr after feeding. A dose response to injected egg development neurosecretory hormone (EDNH) was observed in vivo that was similar to in vitro dose responses previously reported. Blood-fed decapitated females responded equally well to boiled or unboiled head extract. When blood-fed decapitated females were injected with head extract, ecdysteroid levels increased. Partial purification of head extract using high-pressure liquid chromatography yielded a fraction at 34% acetonitrile that showed egg maturation activity in vivo when injected into blood-fed decapitated females, and ecdysiotropic activity when incubated in vitro with ovaries. In addition, a fraction at 30% acetonitrile was found that showed activity in vivo but not in vitro and may be a precursor. Occasionally, the fraction at 37% acetonitrile showed activity in the in vitro assay but had little activity in vivo and may be a metabolite. These results suggest that the same hormone was being assayed in vivo and in vitro and is EDNH.     

The von Willebrand factor-reducing activity of thrombospondin-1 is located in the calcium-binding/C-terminal sequence and requires a free thiol at position 974

Plasma von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury; however, only the very large VWF multimers are effective in promoting platelet adhesion in flowing blood. The multimeric size of VWF can be controlled by the glycoprotein, thrombospondin-1 (TSP-1), which facilitates reduction of the disulfide bonds that hold VWF multimers together. The TSP family of extracellular glycoproteins consists of 5 members in vertebrates, TSP-1 through TSP-4 and TSP-5/COMP. TSP-1 and TSP-2 are structurally similar trimeric proteins composed of disulfide-linked 150-kDa monomers. Recombinant pieces of TSP-1 and TSP-2 incorporating combinations of domains that span the entire subunit were produced in insect cells and examined for VWF reductase activity. VWF reductase activity was present in the Ca(++)-binding repeats and C-terminal sequence of TSP-1, but not of TSP-2. Alkylation of Cys974 in the C-terminal TSP-1 construct, which is a serine in TSP-2, ablated VWF reductase activity. These results imply that the reductase function of TSP-1 centers around Cys974 in the C-terminal sequence.     

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin

Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575-1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a "feet"-,"body"-, and "head"-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys-His-Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying Cys-His-Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.     

Role for lysosomal enzyme beta-hexosaminidase in the control of mycobacteria infection

The pathogenic mycobacteria that cause tuberculosis (TB) and TB-like diseases in humans and animals elude sterilizing immunity by residing within an intracellular niche in host macrophages, where they are protected from microbicidal attack. Recent studies have emphasized microbial mechanisms for evasion of host defense; less is known about mycobactericidal mechanisms that remain intact during initial infection. To better understand macrophage mechanisms for restricting mycobacteria growth, we examined Mycobacterium marinum infection of Drosophila S2 cells. Among approximately 1,000 host genes examined by RNAi depletion, the lysosomal enzyme beta-hexosaminidase was identified as an important factor in the control of mycobacterial infection. The importance of beta-hexosaminidase for restricting mycobacterial growth during mammalian infections was confirmed in macrophages from beta-hexosaminidase knockout mice. Beta-hexosaminidase was characterized as a peptidoglycan hydrolase that surprisingly exerts its mycobactericidal effect at the macrophage plasma membrane during mycobacteria-induced secretion of lysosomes. Thus, secretion of lysosomal enzymes is a mycobactericidal mechanism that may have a more general role in host defense.     

The bifunctional activity of ubiquinone in lysosomal membranes

Ubiquinone is inhomogenously distributed in subcellular biomembranes. Apart from mitochondria where ubiquinone was demonstrated to exert bioenergetic and pathophysiological functions, unusually high levels of ubiquinone were also reported to exist in Golgi vesicles and lysosomes. In lysosomes the interior differs from other organelles by the low pH-value, which is important not only to arrest proteins but also to ensure optimal activity of hydrolytic enzymes. Since redox-cycling of ubiquinone is associated with the acceptance and release of protons, we assumed that ubiquinone is a part of a redox chain contributing to unilateral proton distribution. A similar function of ubiquinone was earlier suggested by Crane to operate in Golgivesicles. Support for the involvement of ubiquinone in a presumed couple of redox-carriers came from our observation that almost 70% of total lysosomal ubiquinone was in the divalently reduced state. Further reduction was seen in the presence of external NADH. Analysis of the components involved in the transfer of reducing equivalents from cytosolic NADH to ubiquinone revealed the existence of a FAD-containing NADH-dehydrogenase. The latter was found to reduce ubiquinone by means of a b-type cytochrome. Proton translocation into the interior was linked to the activity of the novel lysosomal redox chain. Oxygen was found to be the terminal electron acceptor, thereby also regulating acidification of the lysosomal matrix. In contrast to mitochondrial respiration, oxygen was only trivalently reduced, giving rise to the release of HO^•-radicals. The role of this novel proton-pumping redox chain and the significance of the associated ROS formation has to be elucidated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of 5.8 kDa C-terminal fragments of Alzheimer amyloid p protein precursor generated in the lysosomal system

Elucidation of the normal metabolic pathways of amyloid β protein precursor (AβPP) is one of the important fields in the study of Alzheimer's disease. It has been suggested that the endosomal-lysosomal pathway may play a key role in the metabolism ofAβPP. We prepared different subcellular fractions from rat brain using a discontinuous sucrose density gradient method. The lysosome-enriched fraction was identified morphologically and biochemically. Various antibodies against the C-terminus ofAβPP were employed to detect specific fragments of AβPP in different subcellular fractions by western blot. The bands of APPP fragments with apparent molecular weight of 5.8 kDa, possibly containing the whole cytoplasmic domain of AβPP, were present specifically in the lysosome-enriched fraction. The 5.8 kDa fragments were increased and full-length APPP was decreased during incubation of the lysosome-enriched fraction in acidic buffer. The results provided direct evidence for the degradation of AβPP in the lysosomal system. Our data indicate that the digestion of AβPP into these small peptides might be important in the pathogenesis of Alzheimer's disease.     

Role of the C-terminal domain in inactivation of brain and cardiac sodium channels

Inactivation is a fundamental characteristic of Na(+) channels, and small changes cause skeletal muscle paralysis and myotonia, epilepsy, and cardiac arrhythmia. Brain Na(v)1.2a channels have faster inactivation than cardiac Na(v)1.5 channels, but minor differences in inactivation gate structure are not responsible. We constructed chimeras in which the C termini beyond the fourth homologous domains of Na(v)1.2a and Na(v)1.5 were exchanged. Replacing the C-terminal domain (CT) of Na(v)1.2a with that of Na(v)1.5 (Na(v)1.2/1.5CT) slowed inactivation at +40 mV approximately 2-fold, making it similar to Na(v)1.5. Conversely, replacing the CT of Na(v)1.5 with that of Na(v)1.2a (Nav1.5/1.2CT) accelerated inactivation, making it similar to Na(v)1.2a. Activation properties were unaffected. The voltage dependence of steady-state inactivation of Na(v)1.5 is 16 mV more negative than that of Na(v)1.2a. The steady-state inactivation curve of Na(v)1.2a was shifted +12 mV in Na(v)1.2/1.5CT, consistent with destabilization of the inactivated state. Conversely, Na(v)1.5/1.2CT was shifted -14 mV relative to Na(v)1.5, consistent with stabilization of the inactivated state. Although these effects of exchanging C termini were consistent with their effects on inactivation kinetics, they magnified the differences in the voltage dependence of inactivation between brain and cardiac channels rather than transferring them. Thus, other parts of these channels determine the basal difference in steady-state inactivation. Deletion of the distal half of either the Na(v)1.2 or Na(v)1.5 CTs accelerated open-state inactivation and negatively shifted steady-state inactivation. Thus, the C terminus has a strong influence on kinetics and voltage dependence of inactivation in brain Na(v)1.2 and cardiac Na(v)1.5 channels and is primarily responsible for their differing rates of channel inactivation.     

Role of cathepsins in ovarian follicle growth and maturation

Several complex processes are involved in the production of viable eggs. The aim of this review is to provide an overview on the role played by lysosomal enzymes, especially cathepsins B, D, and L, during ovarian follicle growth and maturation. Specific attention is focused on the relationship between the second proteolytic cleavage of yolk proteins (YP) and the resumption of the meiosis during germinal vesicle break down (GVBD). Maturation represents the final stage of oocytes development prior to ovulation. Oocytes in this phase appear translucent. In many teleosts GVBD is accompanied by water uptake and among marine teleosts with pelagic eggs, most of the final volume is reached by this process. The last phase of maturation in benthonic eggs also occurs concomitant to a second proteolytic cleavage and is related with a slight hydration process. In vitro maturation by 17alpha,20beta-dihydroxy-4-pregnen-3one in class III Danio rerio oocytes, induced 80% of GVBD. The maturation of these oocytes is known to be associated with proteolysis of their major yolk components. In the present study, we show that inhibition of specific enzymes (cathepsins) involved in the second YP processing, did not affect the occurrence of GVBD as the oocytes become translucent and display a slight increase in size. More specifically, in vitro incubation of the maturing oocytes with a cathepsin B inhibitor suppressed both cathepsin B and L activities and the proteolysis of YP. On the contrary, the addition of cathepsin L inhibitor, only affected cathepsin L activity, indicating that cathepsin B is probably involved in Cathepsin L activation, and this enzyme is probably responsible for the second YP processing. These results, together with previous studies, indicate that the GVBD process is independent of the occurrence of the second proteolytic process. It supports the hypothesis that the maturation process is under K+ ion flux control, while yolk proteolysis is related to the temporal and specific activation of cathepsins by acidification of yolk spheres.     

Role of monoamines in the pathogenesis of sex maturation disorders

Serotonin and norepinephrine levels in the hypothalamus and blood of albino female rats with a normal and abnormal course of puberty were studied to find a solution to the problem of the role of monoamines in the pathogenesis of pubertal disorders. Intense physical exercise the 21- and 40-day animals were subjected to, was used to simulate a delay in puberty. It was established that the nature and intensity of disorders of the sexual system were determined, to a certain extent, by changes in the monoamine content in the hypothalamus over time. A significant rise of the serotonin level and a simultaneous decrease in the epinephrine level were revealed in the rats with delayed puberty. Possible mechanisms of the role of monoamines in the pathogenesis of pubertal disorders, function of the female sexual system in the period of puberty are discussed.     

Role of common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of murine mast cells

The regulatory roles of the common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using gamma(c)-deficient (gamma(c)(-)) and Jak3-deficient (Jak3(-)) mice. Although the mast cells in gamma(c)(-) and Jak3(-) mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in gamma(c)(-) and Jak3(-) mice as compared with that in wild-type mice. Among gamma(c)-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow-derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from gamma(c)(-) and Jak3(-) mice. In addition, IL-4Ralpha, gamma(c), and Jak3, but not IL-2Rbeta or IL-7Ralpha, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Ralpha1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from gamma(c)(-) and Jak3(-) mice. These results indicate that gamma(c)- and Jak3-dependent signaling is essential for IL-4- and IL-9-induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a gamma(c)- and Jak3-dependent manner.     

Role of gamma knife radiosurgery in the treatment of prolactinomas

Pituitary - Stereotactic radiosurgery is one of the treatment options for prolactinomas, the most commonly used being Gamma Knife Radiosurgery (GKRS). GKRS is indicated mainly in the treatment of...     

Temporal variations of hemolymph esterase activity and juvenile hormone titers during ovocyte maturation in Acheta domesticus (Orthoptera)

Using in vitro methods, juvenile hormone (JH) esterase activity and alpha-naphthylacetate esterase activity were determined in the hemolymph during the first reproductive cycle of the house cricket, Acheta domesticus. Biochemical properties of the hemolymph JH esterase were studied. alpha-Naphthylacetate esterases increased during the first gonotrophic cycle: peaks of their activity could be observed concomitant with peaks of JH esterase activity. The fluctuations in JH esterase activity correlated with those of hemolymph JH titers. The results are discussed.     

Role of cyclic nucleotide signaling in oocyte maturation

The development of the ovarian follicle, oocyte maturation, and ovulation require a complex set of endocrine, paracrine, and autocrine inputs that are translated into the regulation of cyclic nucleotide levels. Changes in intracellular cAMP mediate the gonadotropin regulation of granulosa and theca cell functions. Likewise, a decrease in cAMP concentration in the oocyte has been associated with the resumption of meiosis. Using pharmacological and molecular approaches, we determined that the expression of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, is compartmentalized in the ovarian follicle of all species studied, with PDE3 present in the oocytes and PDE4s in granulosa cells. The PDE3 expressed in the mouse oocyte was cloned, and the protein expressed in a heterologous system had properties similar to those of a PDE3A derived from somatic cells. Inhibition of the oocyte PDE3 completely blocked oocyte maturation in vitro and in vivo, demonstrating that the activity of this enzyme is essential for oocyte maturation. Heterologous expression of PDE3A in Xenopus oocyte causes morphological changes distinctive of resumption of meiosis (GVBD), as well as activation of mos translation and MAPK phosphorylation. Using mRNA and antibody microinjection in the Xenopus eggs, we have shown that PDE3 is downstream from the kinase PKB/Akt in the pathway that mediates IGF-1 but not progesterone-induced meiotic resumption. The presence of a similar regulatory module in mammalian oocytes is inferred by pharmacological studies with PDE3 inhibitors and measurement of PDE activity. Thus, PDE3 plays an essential role in the signaling pathway that controls resumption of meiosis in amphibians and mammals. Understanding the regulation of this enzyme may shed some light on the signals that trigger oocyte maturation.