Metalloendoprotease inhibitors block protein synthesis, intracellular transport, and endocytosis in hepatoma cells

Fusion of membrane vesicles has been implicated in many intracellular processes including the transport of proteins destined for secretion or storage. Vesicular transport coupled with membrane fusion has been demonstrated for rough endoplasmic reticulum to Golgi and Golgi to plasma membrane transport as well as receptor mediated endocytosis and receptor recycling. Recent studies with inhibitors suggest that metalloendoproteases may mediate a wide variety of intracellular fusion events. Thus, in order to examine the potential role of metalloendoproteases in both transport/secretion and endocytosis/recycling we have used selected dipeptide substrates to probe these processes in human HepG2 cells. Using pulse-chase labeling, immunoprecipitation, and polyacrylamide gel electrophoresis we show that transport and secretion of newly synthesized proteins along the exocytotic route were completely inhibited by substrate dipeptides (e.g. Cbz-Gly-Phe-amide, where Cbz is benzyloxycarbonyl) but not by irrelevant dipeptides (e.g. Cbz-Gly-Gly-amide). The effect was rapid, reversible, and specific. The secretory pathway was blocked between the rough endoplasmic reticulum and Golgi as well as Golgi and plasma membrane as judged by the status of N-glycosylation intermediates. In addition, these inhibitors specifically inhibited protein synthesis without alterations in cellular ATP concentrations. However, cell-free amino acid incorporation was not inhibited. Receptor-mediated uptake of asialoglycoproteins was specifically and reversibly inhibited by dipeptide substrates. This effect appears to be secondary to inhibition of recycling as neither ligand binding nor internalization were affected. Thus the present observations suggest that metalloendoprotease activity may be involved in the regulation of multiple intracellular pathways perhaps at the level of vesicular fusion events.     

Possible involvement of poly(A) in protein synthesis.

The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect.     

Co-expression for intracellular processing in microbial protein production

The biological activity of a recombinant protein is highly dependent on its biophysical properties including post-translational modifications, solubility, and stability. Production of active recombinant proteins requires careful design of the expression strategy and purification schemes. This is often achieved by proper modification of the target protein during and/or after protein synthesis in the host cells. Such co-translational or post-translational processing of recombinant proteins is typically enabled by co-expressing the required enzymes, folding chaperones, co-factors and/or processing enzymes in the host. Various applications of the co-expression technology in protein production are discussed in this review with representative examples described.     

Amyloid precursor-like protein 1 influences endocytosis and proteolytic processing of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.     

Axonal amyloid precursor protein and its fragments undergo somatodendritic endocytosis and processing

Deposition of potentially neurotoxic Aβ fragments derived from amyloid precursor protein (APP) at synapses may be a key contributor to Alzheimer''s disease. However, the location(s) of proteolytic processing and subsequent secretion of APP fragments from highly compartmentalized, euploid neurons that express APP and processing enzymes at normal levels is not well understood. To probe the behavior of endogenous APP, particularly in human neurons, we developed a system using neurons differentiated from human embryonic stem cells, cultured in microfluidic devices, to enable direct biochemical measurements from axons. Using human or mouse neurons in these devices, we measured levels of Aβ, sAPPα, and sAPPβ secreted solely from axons. We found that a majority of the fragments secreted from axons were processed in the soma, and many were dependent on somatic endocytosis for axonal secretion. We also observed that APP and the β-site APP cleaving enzyme were, for the most part, not dependent on endocytosis for axonal entry. These data establish that axonal entry and secretion of APP and its proteolytic processing products traverse different pathways in the somatodendritic compartment before axonal entry.     

Possible involvement of transglutaminase in endocytosis and antigen presentation

Experiments were carried out to determine as to whether or not internalization of antigen is necessary for subsequent antigen presentation by accessory cells using monoamines which are known as transglutaminase (TGase) inhibitors. It was found that endocytosis for immune complexes via Fc receptors such as sheep erythrocytes coated with IgG class antibody (EA) was different from receptor-independent endocytosis for soluble protein such as horse radish peroxidase (HRP) in the sensitivity to monoamines; methylamine inhibited the receptor-dependent endocytosis of immune complexes at a concentration of over 20 mM and the receptor-independent endocytosis of HRP at 2 mM, while dansylcadaverine (DC) inhibited both at a concentration of 100 microM. It was noteworthy that antigen-specific T cell proliferation to splenic adherent cells pulsed with DNP9.6-ovalbumin (DNP9.6-OVA) was blocked strongly by DC as well, but weakly by methylamine. These results suggest the possibility that antigen presentation requires internalization of antigen by a mechanism such as receptor-dependent endocytosis for the subsequent reexpression of antigen on membranes. Furthermore, it was confirmed that TGase activity is high in peritoneal exudate and spleen adherent cells, both of which have accessory cell activities for lymphocytes, suggesting the possibility that TGase might be involved intimately in receptor-dependent endocytosis and subsequent antigen presentation.     

The roles of RNA in the synthesis of protein

The crystal structures of ribosomes that have been obtained since 2000 have transformed our understanding of protein synthesis. In addition to proving that RNA is responsible for catalyzing peptide bond formation, these structures have provided important insights into the mechanistic details of how the ribosome functions. This review emphasizes what has been learned about the mechanism of peptide bond formation, the antibiotics that inhibit ribosome function, and the fidelity of decoding.     

Possible involvement of messenger RNA-associated proteins in protein synthesis

Two distinct forms of globin messenger RNA were isolated from mouse spleen cells infected with Friend erythroleukemia virus: polyribosomal messenger ribonucleoprotein particles (15S mRNP), and their corresponding protein-free mRNAs obtained by chemical deproteinization. The translation efficiencies of both messenger forms were assayed in a Krebs II ascites cell-free system. Selective removal of RNA-binding proteins from the ascites cell lysate did not affect globin synthesis when the mRNA was supplied as 15S mRNP; deproteinized mRNA however was not translated. Only in the presence of two fractions of RNA-binding proteins was the protein-free mRNA translated. Some of the RNA-binding proteins have the same molecular weights and isoelectric points as the principal proteins of 15S mRNP.     

Roles of protein synthesis and tRNA aminoacylation in the regulation of intracellular protein breakdown in E. coli

The previously suggested roles of protein synthesis and tRNA aminoacylation in the regulation of intracellular protein breakdown were examined in strains of E. coli temperature-sensitive for aminoacyl-tRNA synthetases. Direct measurements of tRNA aminoacylation show no correlation between the degree of tRNA charging and the rate of protein breakdown. Protein breakdown was accelerated by transfer from 30°C to 42°C to about the same degree in temperature-sensitive mutants as in related normal strains. Deprivation of inorganic phosphate at the high temperature stimulated further protein breakdown in normal, but not in temperature-sensitive strains. It is concluded that the regulation of protein breakdown requires concomitant protein synthesis and is not influenced by the level of aminoacylation of tRNA.     

Isoprenoid requirement for intracellular transport and processing of murine leukemia virus envelope protein.

Lovastatin blocks the biosynthesis of the isoprenoid precursor, mevalonate. When Friend murine erythroleukemia (MEL) cells are cultured in medium containing lovastatin, the precursor of murine leukemia virus envelope glycoprotein (gPr90env) fails to undergo proteolytic processing, which normally occurs in the Golgi complex. Consequently, newly synthesized envelope proteins are not incorporated into viral particles that are shed into the culture medium. gPr90env appears to be localized in a pre-Golgi membrane compartment, based on its enrichment in subcellular fractions containing NADPH-cytochrome c reductase activity and the sensitivity of its carbohydrate chains to digestion with endoglycosidase H. Arrest of gPr90env processing occurs at concentrations of lovastatin that are not cytostatic, and the effect of the inhibitor is prevented by addition of mevalonate to the medium. The low molecular mass GTP-binding proteins, rab1p and rab6p, which are believed to function in early steps of the exocytic pathway, are normally modified posttranslationally by geranylgeranyl isoprenoids. However, in MEL cells treated with 1 microM lovastatin, nonisoprenylated forms of these proteins accumulate in the cytosol prior to arrest of gPr90env processing. These observations suggest that lovastatin may prevent viral envelope precursors from reaching the Golgi compartment by blocking the isoprenylation of rab proteins required for ER to Golgi transport.     

Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000-68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000-Mr form by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post-translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth-surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus. Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.     

Dissociation of functional roles of dynamin in receptor-mediated endocytosis and mitogenic signal transduction.

Dynamin plays a critical role in the membrane fission mechanism that mediates regulated endocytosis of many G protein-coupled receptors. In addition, dynamin is required for ligand-induced activation of mitogen-activated protein kinase by certain receptors, raising a general question about the role of dynamin in mitogenic signal transduction. Here we report that endocytosis of mu and delta opioid receptors is not required for efficient ligand-induced activation of mitogen-activated protein kinase. Nevertheless, mitogenic signaling mediated by these receptors is specifically dynamin-dependent. Thus a functional role of dynamin in mitogenic signaling can be dissociated from its role in receptor-mediated endocytosis, suggesting a previously unidentified and distinct role of dynamin in signal transduction by certain G protein-coupled receptors.     

Mechanisms and cellular roles of local protein synthesis in mammalian cells

After the export from the nucleus it turns out that all mRNAs are not treated equally. Not only is mRNA subject to translation, but also through RNA-binding proteins and other trans-acting factors, eukaryotic cells interpret codes for spatial sorting within the mRNA sequence. These codes instruct the cytoskeleton and translation apparatus to make decisions about where to transport and when to translate the intended protein product. Signaling pathways decode extra-cellular cues and can modify transport and translation factors in the appropriate cytoplasmic space to achieve translation locally. Identifying regulatory sites on transport factors as well as novel physiological functions for well-known translation factors has provided significant advances in how spatially controlled translation impacts cell function.     

Inhibition of protein synthesis stimulates intracellular protein degradation in growing yeast cells

In exponentially growing cells of Saccharomyces cerevisiae, cycloheximide stimulated intracellular protein degradation to the same extent as did starvation for required amino acids. By using inhibitors of macromolecular synthesis and temperature-sensitive mutants defective in different steps of RNA and protein synthesis it could be demonstrated, that this stimulation of protein degradation was directly related to the inhibition of protein synthesis per se, but not connected to the cessation of ribosomal RNA synthesis or to the inhibition of cell growth.     

Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis

The ATP-dependent dissociation of clathrin from clathrin-coated vesicles (CCVs) by the molecular chaperone Hsc70 requires J-domain cofactor proteins, either auxilin or cyclin-G-associated kinase (GAK). Both the nerve-specific auxilin and the ubiquitous GAK induce CCVs to bind to Hsc70. The removal of auxilin or GAK from various organisms and cells has provided definitive evidence that Hsc70 uncoats CCVs in vivo. In addition, evidence from various studies has suggested that Hsc70 and auxilin are involved in several other key processes that occur during clathrin-mediated endocytosis. First, Hsc70 and auxilin are required for the clathrin exchange that occurs during coated-pit invagination and constriction; this clathrin exchange may catalyze any rearrangement of the clathrin-coated pit (CCP) structure that is required during invagination and constriction. Second, Hsc70 and auxilin may chaperone clathrin after it dissociates from CCPs so that it does not aggregate in the cytosol. Third, auxilin and Hsc70 may be involved in the rebinding of clathrin to the plasma membrane to form new CCPs and independently appear to chaperone adaptor proteins so that they can also rebind to membranes to nucleate the formation of new CCPs. Finally, if formation of the curved clathrin coat induces membrane curvature, then Hsc70 and auxilin provide the energy for this curvature by inducing ATP-dependent clathrin exchange and rearrangement during endocytosis and ATP-dependent dissociation of clathrin at the end of the cycle so that it is energetically primed to rebind to the plasma membrane.     

Protein kinase C-zeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting

We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-zeta (PKCzeta) expression and by overexpression of a dominant negative PKCzeta mutant (DN-PKCzeta). Conversely, overexpression of PKCzeta increased insulin internalization as a function of the PKCzeta levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-alpha or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKCzeta inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKCzeta bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKCzeta appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.