Intracellular Turnover Research Articles

Theoretical and experimental quantum efficiencies of the growth of anoxygenic phototrophic bacteria

This work is devoted to investigation of regularities of mass–energy balance of purple bacteria photorophic growth. Estimates of culture growth efficiency were derived from bioenergetic properties of cell metabolism. An equation system describing joint balance of flows of metabolite reductivity, high-energy protons (HEP) and high-energy bonds (HEB) was formulated. The maximum cell yield from light and the rate of energy expenditure for cell maintenance were found as mathematical expressions containing metabolic stoichiometric coefficients, rates of energy losses and intracellular matter turnover. Experimental study was conducted with a purple bacterium Rhodobacter capsulatus grown continuously on hydrogen at different illumination intensities. The obtained numerical estimates of attainable maximum growth yield were compared with experimental data both taken from literature and obtained in this work. Theoretical and experimental yield values showed good agreement.

Exogenous cadherin microdisplay can interfere with endogenous signaling and reprogram gene expression in cultured hepatocytes

We recently found that the basal micro substrate presentation of E-cadherin, a key cell-cell adhesion molecule in the liver, can modulate hepatocellular proliferative potential and differentiated function (Brieva and Moghe, in press). In the current study, we established a similar experimental model involving rat hepatocytes cultured on collagen and incorporated 5 microm polystyrene microbeads functionalized with Protein A-anchored E-cadherin/human lgG Fc chimeric fusion constructs. We investigated the cadherin governed dose-response of cell proliferative potential and quantified the underlying changes in intracellular gene signaling processes. Hepatocellular proliferative potential was found to be intensified with an increase in the microdisplay of acellular cadherins and this effect was offset by increased cell seeding density. Notably, we report that following overnight exposure to acellular cadherins, the expression of genes known to mediate the control of cell proliferation, cyclin D1 and c-myc, was upregulated, while the expression of differentiation-related genes, namely albumin and cytochrome p450 II B1, was reduced. The exposure of cell cultures to exogenous cadherins was found to markedly disrupt the localization of endogenous E-cadherin and beta-catenin to junctions at cell-cell contacts and cause a quantitative decrease in the endogenous cadherin protein levels. Based on all of our observations, we propose that the acellular presentation of E-cadherin chimeras competitively disrupts endogenous cadherin containing complexes at cell-cell junctions and increases intracellular cadherin turnover, thereby promoting beta-catenin mediated signaling, which ultimately engenders an increase in cell proliferative potential and a decrease in differentiated function.

Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation.

The exocytic pathway provides a physical route through which newly synthesized secretory and membrane proteins are deployed to the eukaryote cell surface. For newly synthesized alpha1-antitrypsin (AAT), the modification of its asparagine-linked oligosaccharides by a slow-acting mannosidase partitions the misfolded monomer into the proteasomal degradation pathway. Herein, we asked whether, and how, modification by endoplasmic reticulum mannosidase I (ERManI) contributes to the preferential selection of the misfolded AAT monomer for proteasomal degradation. Transiently expressed mutant and WT AAT variants underwent rapid destabilization in response to an artificially elevated ERManI concentration in the murine hepatoma cell line, Hepa1a. Based on the mannosidase- and lactacystin-sensitive properties of intracellular turnover, a stochastic model is proposed in which the delayed onset of the glycan modification, relative to the duration of nonnative protein structure, coordinates the preferential degradation of the misfolded monomer and spares the native molecule from destruction. Newly synthesized endogenous transferrin underwent degradation in response to an elevated concentration of ERManI, whereas the nonglycosylated secretory glycoprotein albumin was not affected. Taken together, these findings indicate that efficient conformational maturation might function as the initial quality control standard for a broad population of glycoproteins.

Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells.

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that approximately 68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of approximately 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.

Stimulatory effect of vitamin C on autophagy in glial cells.

Intracellular accumulation of damaged or abnormal proteins is a common event associated with numerous neurodegenerative diseases and other age-related pathologies. Increasing the activity of the intracellular proteolytic systems normally responsible for the removal of these abnormal proteins might be beneficial in lessening the severity or development of those pathologies. In this study we have used human astrocyte glial cells to investigate the effect of vitamin C (ascorbate) on the intracellular turnover of proteins. Supplementation of the culture medium with physiological concentrations of vitamin C did not affect protein synthesis, but did increase the rate of protein degradation by lysosomes. Vitamin C accelerated the degradation of intra- and extracellular proteins targeted to the lysosomal lumen by autophagic and heterophagic pathways. At the doses analyzed, vitamin C lowered and stabilized the acidic intralysosomal pH at values that result in maximum activation of the lysosomal hydrolases.

Influence of Peroxisome Proliferator-activated Receptor α Agonists on the Intracellular Turnover and Secretion of Apolipoprotein (Apo) B-100 and ApoB-48

The peroxisome proliferator-activated receptor (PPAR) alpha agonist WY 14,643 increased the secretion of apolipoprotein (apo) B-100, but not that of apoB-48, and decreased triglyceride biosynthesis and secretion from primary rat hepatocytes. These effects resulted in decreased secretion of apoB-100-very low density lipoprotein (VLDL) and an increased secretion of apoB-100 on low density lipoproteins/intermediate density lipoproteins. ApoB-48-VLDL was also replaced by more dense particles. The proteasomal inhibitor lactacystin did not influence the recovery of apoB-100 or apoB-48 in primary rat hepatocytes, indicating that co-translational (proteasomal) degradation is of less importance in these cells. Treatment with WY 14,643 made the recovery of apoB-100 sensitive to lactacystin, most likely reflecting the decreased biosynthesis of triglycerides. The PPAR alpha agonist induced a significant increase in the accumulation of pulse-labeled apoB-100 even after a short pulse (2-5 min). There was also an increase in apoB-100 nascent polypeptides, indicating that the co-translational degradation of apoB-100 was inhibited. However, a minor influence on an early posttranslation degradation cannot be excluded. This decreased co-translational degradation of apoB-100 explained the increased secretion of the protein. The levels of apoB-48 remained unchanged during these pulse-chase experiments, and albumin production was not affected, indicating a specific effect of PPAR alpha agonists on the co-translational degradation of apoB-100. These findings explain the difference in the rate of secretion of the two apoB proteins seen after PPAR alpha activation. PPAR alpha agonists increased the expression and biosynthesis of liver fatty acid-binding protein (LFABP). Increased expression of LFABP by transfection of McA-RH7777 cells increased the secretion of apoB-100, decreased triglyceride biosynthesis and secretion, and increased PPAR alpha mRNA levels. These findings suggest that PPAR alpha and LFABP could interact to amplify the effect of endogenous PPAR alpha agonists on the assembly of VLDL.

Rapid biologically mediated oxygen isotope exchange between water and phosphate

In order to better constrain the rate of oxygen isotope exchange between water and phosphate via biochemical reactions a set of controlled experiments were conducted in 1988 at the Aquaculture Plant in Elat, Israel. Different species of algae and other organisms were grown in seawater tanks under controlled conditions, and the water temperature and oxygen isotopic composition (δ18Ow) were monitored. The oxygen isotopic composition of phosphate (δ18Op) in the organisms' food source, tissues, and the δ18Op of dissolved inorganic phosphate (DIP) were measured at different stages of the experiments. Results indicate that intracellular oxygen isotope exchange between phosphorus compounds and water is very rapid and occurs at all levels of the food chain. Through these reactions the soft tissue δ18Op values become 23–26‰ higher than δ18Ow, and δ18Op values of DIP become ∼20‰ higher than δ18Ow. No correlation between δ18Op values and either temperature or P concentrations in these experiments was observed. Our data imply that biogenic recycling and intracellular phosphorus turnover, which involves kinetic fractionation effects, are the major parameters controlling the δ18Op values of P compounds dissolved in aquatic systems. This information is fundamental to any application of δ18Op of dissolved organic or inorganic phosphate to quantify the dynamics of phosphorus cycling in aquatic systems.

Exogenous interleukin-2 administration corrects the cell cycle perturbation of lymphocytes from human immunodeficiency virus-infected individuals.

Human immunodeficiency virus (HIV)-induced immunodeficiency is characterized by progressive loss of CD4(+) T cells associated with functional abnormalities of the surviving lymphocytes. Increased susceptibility to apoptosis and loss of proper cell cycle control can be observed in lymphocytes from HIV-infected individuals and may contribute to the lymphocyte dysfunction of AIDS patients. To better understand the relation between T-cell activation, apoptosis, and cell cycle perturbation, we studied the effect of exogenous interleukin-2 (IL-2) administration on the intracellular turnover of phase-dependent proteins. Circulating T cells from HIV-infected patients display a marked discrepancy between a metabolic profile typical of G(0) and a pattern of expression of phase-dependent proteins that indicates a more-advanced position within the cell cycle. This discrepancy is enhanced by in vitro activation with ConA and ultimately results in a marked increase of apoptotic events. Conversely, treatment of lymphocytes with IL-2 alone restores the phase-specific pattern of expression of cell cycle-dependent proteins and is associated with low levels of apoptosis. Interestingly, exogenous IL-2 administration normalizes the overall intracellular protein turnover, as measured by protein synthesis, half-life of newly synthesised proteins, and total protein ubiquitination, thus providing a possible explanation for the effect of IL-2 on the intracellular kinetics of cell cycle-dependent proteins. The beneficial effect of IL-2 administration is consistent with the possibility of defective IL-2 function in vivo, which is confirmed by the observation that lymphocytes from HIV-infected patients show abnormal endogenous IL-2 paracrine/autocrine function upon in vitro mitogen stimulation. Overall these results confirm that perturbation of cell cycle control contributes to HIV-related lymphocyte dysfunction and, by showing that IL-2 administration can revert this perturbation, suggest a new mechanism of action of IL-2 therapy in HIV-infected patients.

Intracellular trafficking and turnover of phosphatidylinositol 3-phosphate

Phosphatidylinositol 3-kinases (PI 3-kinases) regulate cellular functions through the 3′ -phosphorylation of phosphatidylinositol (PI) and its derivatives. The PI 3-kinase product phosphatidylinositol 3-phosphate [PI(3)P] functions to recruit and activate effector proteins containing FYVE zinc finger domains. These proteins have various functions in endocytic membrane trafficking, cytoskeletal regulation and signal transduction. In order to understand the function of FYVE proteins, it is essential to study the formation, localisation, trafficking and turnover of PI(3)P. Here we review recent evidence that PI(3)P is formed on early endosomes through the activity of a PI 3-kinase which is recruited by the GTPase Rab5, and that the PI(3)P is subsequently internalised into intralumenal vesicles of multivesicular endosomes for turnover.

Abnormal intracellular kinetics of cell-cycle–dependent proteins in lymphocytes from patients infected with human immunodeficiency virus: a novel biologic link between immune activation, accelerated T-cell turnover, and high levels of apoptosis

Human immunodeficiency virus (HIV)-infection is characterized by loss of CD4+ T cells associated with high levels of immune activation, T-cell proliferation, and lymphocyte apoptosis. To investigate the role of intrinsic perturbations of cell-cycle control in the immunopathogenesis of acquired immunodeficiency syndrome (AIDS), we studied the expression of cell-cycle-dependent proteins in lymphocytes from HIV-infected patients. Cyclin B1 expression, Nucleolar Organizer Regions (NORs) number, and NORs area of distribution were all consistently increased in HIV-infected patients, but returned to normal after effective antiretroviral therapy, suggesting that viral replication is directly implicated in the genesis of the observed changes. Analysis of cyclin B1 intracellular turnover showed that the increased cyclin B1 expression is (1) caused by defective degradation in the presence of normal rates of synthesis, and (2) is temporally associated with decreased levels of ubiquitination. After in vitro activation of lymphocytes from healthy individuals, cyclin B1 and cdc25 expression and ubiquitination, p34 cdc2 activity, NORs morphology, and C23/nucleolin localization showed a 72- to 96-hour cyclic pattern that led to a biologic state similar to baseline. On the contrary, complex but consistent changes of the same indices followed activation of T lymphocytes from HIV-infected patients, resulting in a 5-fold increase in apoptosis. Overall, our data indicate that a profound dysregulation of cell-cycle control is present in lymphocytes from HIV-infected patients. This finding may provide a novel biologic link between immune activation, accelerated lymphocyte turnover, and increased apoptosis during HIV infection.

Correction for vol. 102, p. 2700; vol. 102, p. 3053

HomeCirculationVol. 103, No. 3 Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citations ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUB Originally published23 Jan 2001https://doi.org/10.1161/01.CIR.103.3.476Circulation. 2001;103:476This article corrects the followingLimitation of Excessive Extracellular Matrix Turnover May Contribute to Survival Benefit of Spironolactone Therapy in Patients With Congestive Heart Failure In the article, “Limitation of Excessive Intracellular Matrix Turnover May Contribute to Survival Benefit of Spironolactone Therapy in Patients With Congestive Heart Failure: Insights from the Randomized Aldactone Evaluation Study (RALES),” by Zannad et al that appeared in a previous issue of the journal (Circulation. 2000;102:2700–2706 ), some sentences contained incorrect information.In Results, under Baseline Levels of ECM Turnover Markers (page 2702), the sentence: “However, ischemic heart disease (5.3 versus 4.7 μg/L for ischemic versus nonischemic, P=0.02) and baseline treatment with digoxin (4.7 versus 5.4 μg/L for on digoxin versus off digoxin, P=0.001) were independently associated with baseline PIIINP levels” should be replaced with the following:However, ischemic heart disease (5.3 versus 4.7 μg/L for ischemic versus nonischemic, P=0.02) and baseline treatment with digoxin (5.4 versus 4.7 μg/L for on digoxin versus off digoxin, P=0.001) were independently associated with baseline PIIINP levels.In addition, under Prognostic Significance of Serum Levels of ECM Turnover in the Discussion (page 2705), the statement, “On the other hand, we found that patients chronically treated with digitalis had a lower level of PIIINP. This is consistent with the finding that digoxin reduces death due to the progression of heart disease” should be replaced with the following:On the other hand, we found that patients chronically treated with digitalis had a higher level of PIIINP. This is inconsistent with the finding that digoxin reduces death due to the progression of heart disease.In the article by Nelson et al that appeared in a previous issue of the journal (Circulation. 2000;102:3053–3059), the order of authors in the author line was incorrect. The correct author list follows:Gregory S. Nelson, PhD; Ronald D. Berger, MD, PhD; Barry J. Fetics, MSE; Maurice Talbot, RN; Julio C. Spinelli, PhD; Joshua M. Hare, MD; David A. Kass, MD Previous Back to top Next FiguresReferencesRelatedDetailsCited By Bakris G, Vassalotti J, Ritz E, Wanner C, Stergiou G, Molitch M, Nesto R, Kaysen G and Sowers J (2010) National Kidney Foundation consensus conference on cardiovascular and kidney diseases and diabetes risk: an integrated therapeutic approach to reduce events, Kidney International, 10.1038/ki.2010.292, 78:8, (726-736), Online publication date: 1-Oct-2010. Williams E, Katholi R and Karambelas M (2006) Use and side-effect profile of spironolactone in a private cardiologist's practice, Clinical Cardiology, 10.1002/clc.4960290405, 29:4, (149-153), Online publication date: 1-Apr-2006. Related articlesLimitation of Excessive Extracellular Matrix Turnover May Contribute to Survival Benefit of Spironolactone Therapy in Patients With Congestive Heart Failure Faiez Zannad, et al. Circulation. 2000;102:2700-2706 January 23, 2001Vol 103, Issue 3 Advertisement Article InformationMetrics Copyright © 2001 by American Heart Associationhttps://doi.org/10.1161/01.CIR.103.3.476 Originally publishedJanuary 23, 2001 PDF download Advertisement

Peptidases play an important role in cataractogenesis: an immunohistochemical study on lenses derived from Shumiya cataract rats.

The role of proteolytic enzymes in Shumiya cataract rats in alterations to lens proteins during cataract formation was studied immunohistochemically using antibodies against exopeptidases, such as lysosomal dipeptidyl peptidase II (DPP II), cytosolic dipeptidyl peptidase III, and soluble and membrane-bound alanyl aminopeptidases, and against cytosolic endopeptidases such as mu- and m-calpains, and 20S proteasome. AlphaB-crystallin was detected as a proteolytic marker in the lenses. A constant immunoreactivity against all the antibodies employed was observed in the lens epithelium independent of the strain and age of the rats. A weak immunoreactivity against exo- and endopeptidases and an intense reactivity against alphaB-crystallin were observed in the lens fibres of control rats at all ages. The immunoreactivity of these peptidases in lens fibres increased with age in cataract rats, but that of alphaB-crystallin decreased. No reactivity against exo- and endopeptidases was seen in the perinuclear region of lenses of control rats at all ages or in Shumiya cataract rats at 8 and 10 weeks of age, but an intense reactivity against these peptidases was observed in the lens perinuclear region of lenses in cataract rats at 12 and 14 weeks of age. AlphaB-crystallin immunoreactivity was observed with ordered striations in the lens perinuclear region of all control rats whereas the striations in this area of cataract rat lens were disorganized. Membrane-bound alanyl aminopeptidase was detected feebly in the lens epithelium and fibres of both types of rat at all weeks of age. These findings indicate that exo- and endopeptidases, except for membrane-bound alanyl aminopeptidase, are expressed intensively and are age-dependent. Conversely, the amount of alphaB-crystallin decreased with age in lens fibres of cataract rats. Calpains (mu- and m-), 20S proteasome, dipeptidyl peptidases II and III and soluble alanyl aminopeptidase are thought to induce lens opacification kinetically during cataract formation in Shumiya cataract rats through the intracellular turnover of lens proteins.

Fc receptor mediated endocytosis of small soluble immunoglobulin G immune complexes in Kupffer and endothelial cells from rat liver.

Soluble circulating immunoglobulin G immune complexes are mainly eliminated by the liver, predominantly by uptake in the Kupffer cells, but also the liver endothelial cells seem to be of importance. In the present study we have followed the intracellular turnover of immune complexes after Fc(gamma) receptor mediated endocytosis in cultured rat liver endothelial cells and Kupffer cells by means of isopycnic centrifugation, DAB cross-linking and morphological techniques. For the biochemical experiments the antigen, dinitrophenylated bovine serum albumin (BSA), was labeled with radioiodinated tyramine cellobiose that cannot cross biological membranes and therefore traps labeled degradation products at the site of formation. The endocytic pathway followed by immune complexes was compared with that followed by scavenger receptor ligands, such as formaldehyde treated BSA and dinitrophenylated BSA, and the mannose receptor ligand ovalbumin. Both Kupffer cells and liver endothelial cells took up and degraded the immune complexes, but there was a clear delay in the degradation of immune complexes as compared to degradation of ligands taken up via scavenger receptors. The kinetics of the endocytosis of scavenger receptor ligand was unaffected by simultaneous uptake of immune complexes. Experiments using both biochemical and morphological techniques indicated that the delayed degradation was due to a late arrival of the immune complexes at the lysosomes, which partly was explained by retroendocytosis of immune complexes. Electron microscopy studies revealed that the immune complexes were retained in the early endosomes that remained accessible to other endocytic markers such as ovalbumin. In addition, the immune complexes were seen in multivesicular compartments apparently devoid of other endocytic markers. Finally, the immune complexes were degraded in the same lysosomes as the ligands of scavenger receptors. Thus, immune complexes seem to follow an endocytic pathway that is kinetically or maybe morphologically different from that followed by scavenger and mannose receptor ligands.

DNA vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial immunity.

Genetic immunization is a promising new technology for developing vaccines against tuberculosis that are more effective. In the present study, we evaluated the effects of intracellular turnover of antigens expressed by DNA vaccines on the immune response induced by these vaccines in a mouse model of pulmonary tuberculosis. The mycobacterial culture filtrate protein MPT64 was expressed as a chimeric protein fused to one of three variants of the ubiquitin protein (UbG, UbA, and UbGR) known to differentially affect the intracellular processing of the coexpressed antigens. Immunoblot analysis of cell lysates of in vitro-transfected cells showed substantial differences in the degradation rate of ubiquinated MPT64 (i.e., UbG64 < UbA64 < UbGR64). The specific immune response generated in mice correlated with the stability of the ubiquitin-conjugated antigen. The UbA64 DNA vaccine induced a weak humoral response compared to UbG64, and a mixed population of interleukin-4 (IL-4)- and gamma interferon (IFN-gamma)-secreting cells. Vaccination with the UbGR64 plasmid generated a strong Th1 cell response (high IFN-gamma, low IL-4) in the absence of a detectable humoral response. Aerogenic challenge of vaccinated mice with Mycobacterium tuberculosis indicated that immunization with both the UbA64- and UbGR64-expressing plasmids evoked an enhanced protective response compared to the vector control. The expression of mycobacterial antigens from DNA vaccines as fusion proteins with a destabilizing ubiquitin molecule (UbA or UbGR) shifted the host response toward a stronger Th1-type immunity which was characterized by low specific antibody levels, high numbers of IFN-gamma-secreting cells, and significant resistance to a tuberculous challenge.

Relaxation of human ureteral smooth muscle in vitro by modulation of cyclic nucleotide-dependent pathways.

Phosphodiesterases (PDE) are key enzymes regulating intracellular cyclic nucleotide turnover and, thus, smooth muscle tension. Recent reports have indicated the presence of PDE isoenzymes 1, 2, 4, and 5 in cytosolic supernatants prepared from human ureteral smooth muscle homogenates and the ability of second-generation inhibitors of PDE 3, 4, and 5 to relax KCl-induced tension of human ureteral muscle in vitro. The aim of the present study was to evaluate the functional effects of recently developed, third-generation isoenzyme-selective PDE inhibitors, the nitric oxide (NO)-donating agents sodium nitroprusside (SNP) and dihydropyridine (DHP), which is also described as an antagonist of L-type calcium channels, and the adenylyl cyclase-stimulating drug forskolin on tissue tension and cyclic nucleotide levels of human ureteral smooth muscle segments in vitro. Relaxant responses of human ureteral smooth muscle were investigated in vitro using the organ bath technique. Cyclic nucleotides cAMP and cGMP were determined by specific radioimmunoassay following time and dose-dependent incubation of the ureteral tissue with the drugs. The most pronounced relaxing effects on KCl-induced tension of ureteral smooth muscle were exerted by nitrovasodilator SNP, PDE4 inhibitor rolipram, and PDE5 inhibitors E 4021 and morpholinosulfonyl-pyrazolopyrimidine (MSPP). Relaxing potency of the drugs was paralleled by their ability to elevate intracellular levels of cGMP and cAMP, respectively. Our data suggest the possibility of using selective inhibitors of PDE isoenzymes 4 and 5 in the treatment of ureteral stones and ureteral colic.

The Cytoplasmic, Transmembrane, and Stem Regions of Glycosyltransferases Specify Their in Vivo Functional Sublocalization and Stability in the Golgi

We provide evidence for the presence of targeting signals in the cytoplasmic, transmembrane, and stem (CTS) regions of Golgi glycosyltransferases that mediate sorting of their intracellular catalytic activity into different functional subcompartmental areas of the Golgi. We have constructed chimeras of human alpha1, 3-fucosyltransferase VI (FT6) by replacement of its CTS region with those of late and early acting Golgi glycosyltransferases and have stably coexpressed these constructs in BHK-21 cells together with the secretory reporter glycoprotein human beta-trace protein. The sialyl Lewis X:Lewis X ratios detected in beta-trace protein indicate that the CTS regions of the early acting GlcNAc-transferases I (GnT-I) and III (GnT-III) specify backward targeting of the FT6 catalytic domain, whereas the CTS region of the late acting human alpha1,3-fucosyltransferase VII (FT7) causes forward targeting of the FT6 in vivo activity in the biosynthetic glycosylation pathway. The analysis of the in vivo functional activity of nine different CTS chimeras toward beta-trace protein allowed for a mapping of the CTS donor glycosyltransferases within the Golgi/trans-Golgi network: GnT-I < (ST6Gal I, ST3Gal III) < GnT-III < ST8Sia IV < GalT-I < (FT3, FT6) < ST3Gal IV < FT7. The sensitivity or resistance of the donor glycosyltransferases toward intracellular proteolysis is transferred to the chimeric enzymes together with their CTS regions. Apparently, there are at least three different signals contained in the CTS regions of glycosyltransferases mediating: first, their Golgi retention; second, their targeting to specific in vivo functional areas; and third, their susceptibility toward intracellular proteolysis as a tool for the regulation of the intracellular turnover.

Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway.

Upon infection of mammalian cells, Listeria monocytogenes lyses the phagosome and enters the cytosol, where it secretes proteins necessary for its intracellular growth cycle. Consequently, bacterial proteins exposed to the cytosol are potential targets for degradation by host cytosolic proteases. One pathway for degradation of host cytosolic proteins, the N-end rule pathway, involves recognition of the N-terminal amino acid and is mediated by the proteasome. However, very few natural N-end rule substrates have been identified. We have examined the L. monocytogenes ActA protein as a potential target for this pathway. ActA is an essential determinant of L. monocytogenes pathogenesis that is required to induce actin-based motility and cell-to-cell spread. We show that the half-life of a secreted form of ActA can be altered in the mammalian cytosol by changing the N-terminal amino acid. Moreover, the introduction of a destabilizing N-terminus into the functional, surface-bound form of ActA results in a small-plaque phenotype in L2 cells, which is partially reversible by an inhibitor of the proteasome. These results indicate that the L. monocytogenes ActA protein is a natural N-end rule substrate, and that optimal function of ActA in mediating cell-to-cell spread is dependent upon its intracellular turnover rate.

Alpha-MSH can control the essential cofactor 6-tetrahydrobiopterin in melanogenesis.

In the human epidermis both keratinocytes and melanocytes express POMC m-RNA. Immunohistochemical studies of both cell types demonstrate significantly higher levels of alpha-MSH in melanocytes than in keratinocytes. Both cell types also hold the full capacity for de novo synthesis/recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4). 6BH4 is critical for the hydroxylation of the aromatic amino acids L-phenylalanine, L-tyrosine, and L-tryptophan, for nitric oxide production and in various immune modulatory processes. Recently it was shown that tyrosinase activity is regulated by 6BH4 through a specific allosteric inhibition. The tyrosinase/6BH4 inhibition can be activated by 1:1 complex formation between 6BH4 and alpha-MSH, but an excess of alpha-MSH over 6BH4 can inhibit tyrosinase due to complex formation by tyr2 in the alpha-MSH sequence. In both melanocytes and keratinocytes 6BH4 controls the L-tyrosine supply via phenylalanine hydroxylase (PAH). Recently we were able to show that the cellular uptake of L-phenylalanine and its intracellular turnover to L-tyrosine is crucial for melanogenesis. alpha-MSH can promote the production of L-tyrosine via PAH due to activation of the PAH tetramer to the more active dimer by removing 6BH4 from the regulatory binding domain on the enzyme. In conclusion, alpha-MSH can control (1) intracellular L-tyrosine formation from L-phenylalanine in both melanocytes and keratinocytes, and (2) tyrosinase activity, directly, in melanocytes.

The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.

Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31. Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22-31. We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.

Relationship between cytochrome P450 catalytic cycling and stability: fast degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) in hepatoma cells is abolished by inactivation of its electron donor NADPH–cytochrome P450 reductase

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) involved in the metabolism of gluconeogenetic precursors and some cytotoxins is distinguished from other cytochrome P450 enzymes by its rapid turnover (in vivo half-life of 4-7 h), with ligands to the haem iron, both substrates and inhibitors, stabilizing the protein. CYP2E1 is also known to have a high oxidase activity in the absence of substrate, resulting in the production of reactive oxygen radicals. We suggested that the rapid intracellular turnover of the enzyme may be partly due to covalent modifications by such radicals or to other changes during catalytic cycling, in which case the inhibition of electron supply from NADPH-cytochrome P450 reductase would be expected to stabilize the protein. Fao hepatoma cells, where CYP2E1 showed a half-life of 4 h upon serum withdrawal, were treated for 1 h with 0.3 microM diphenylene iodonium (DPI), a suicide inhibitor of flavoenzymes, which resulted in approximately 90% inhibition of the microsomal NADPH-cytochrome P450 reductase and CYP2E1-dependent chlorzoxazone hydroxylase activities. Subsequent cycloheximide chase revealed that the CYP2E1 half-life increased to 26 h. Neither the degradation rates of total protein, CYP2B1 and NADPH-cytochrome P450 reductase nor the cellular ATP level were affected by DPI under the conditions employed. These results demonstrate for the first time that the short half-life of CYP2E1 in vivo may be largely due to the rapid destabilization of the enzyme during catalytic cycling rather than to the intrinsic instability of the protein molecule.