Biomedical subjects

F J van der Wal

Publications and source records attributed to F J van der Wal.

13 recordsLinked to original sources

PubMed2002

The HCMV gene products US2 and US11 target MHC class I molecules for degradation in the cytosol.

Over millions of years of coevolution with their hosts, viruses have developed highly effective strategies to elude the host immune system. The degradation of major histocompatibility complex (MHC) class I heavy chains by human cytomegalovirus (HCMV) is an example of this. Two HCMV proteins, US2 and US11, target newly synthesized MHC class I heavy chains for destruction via a pathway that involves ubiquitin-dependent retrograde transport, or "dislocation", of the heavy chains from the ER to the cytosol, where the proteins are degraded by proteasomes. In this review, US2- and US11-mediated degradation of MHC class I heavy chains is discussed in relation to data concerning the degradation of other ER luminal proteins. A new, unified model for translocon-facilitated dislocation and degradation of MHC class I heavy chains is presented.

Cytomegalovirus↗

PubMed2001

Ubiquitination is essential for human cytomegalovirus US11-mediated dislocation of MHC class I molecules from the endoplasmic reticulum to the cytosol.

Human cytomegalovirus encodes two glycoproteins, US2 and US11, which cause rapid degradation of MHC class I molecules, thus preventing recognition of virus-infected cells by the immune system. This degradation process involves retrograde transport or 'dislocation' of MHC class I molecules from the endoplasmic reticulum (ER) to the cytosol, where they are deglycosylated by an N-glycanase and degraded by the proteasome. At present it is unknown whether ubiquitination is required for US2- and US11-mediated dislocation and degradation of MHC class I molecules. Here, we show that in E36ts20 hamster cells, which contain a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme, US11-mediated degradation of MHC class I molecules is strongly impaired at the non-permissive temperature, indicating the necessity for ubiquitination in this process. We next addressed the question of whether ubiquitination is a condition for the retrograde movement of MHC class I molecules from the ER to the cytosol, or whether ubiquitination is merely required for recognition of dislocated MHC class I molecules by the proteasome. In the absence of a functional ubiquitin system, complexes of US11 and MHC class I molecules accumulate in the ER. In this state the membrane topology of MHC class I molecules does not significantly change, as judged from proteinase K digestions. Thus the results indicate that a functional ubiquitin system is essential for dislocation of MHC class I molecules from the ER to the cytosol.

Animals↗

PubMed1998

Optimization of bacteriocin release protein (BRP)-mediated protein release by Escherichia coli: random mutagenesis of the pCloDF13-derived BRP gene to uncouple lethality and quasi-lysis from protein release.

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli periplasm into the culture medium. However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques. Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures. A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein beta-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium. These BRP derivatives can be more efficiently used for extracellular production of proteins by E. coli than can the original BRP.

Amino Acid Sequence↗

PubMed1997

Interaction of the thiol-dependent reductase ERp57 with nascent glycoproteins.

Calnexin and calreticulin interact specifically with newly synthesized glycoproteins in the endoplasmic reticulum (ER) and function as molecular chaperones. The carbohydrate-specific interactions between ER components and glycoproteins synthesized in isolated canine pancreatic microsomes were analyzed using a cross-linking approach. A carbohydrate-dependent interaction between newly synthesized glycoproteins, the thiol-dependent reductase ERp57, and either calnexin or calreticulin was identified. The interaction between ERp57 and the newly synthesized glycoproteins required trimming of the N-linked oligosaccharide side chain. Thus, it is likely that ERp57 functions as part of the glycoprotein-specific quality control machinery operating in the lumen of the ER.

Animals↗

PubMed1995

The stable bacteriocin release protein signal peptide, expressed as a separate entity, functions in the release of cloacin DF13.

The pCloDF13 encoded bacteriocin release protein (BRP) plays a role in the release of the bacteriocin cloacin DF13. The BRP signal peptide is stable after cleavage, and accumulates in the cytoplasmic membrane. A BRP which is correctly targeted by the unstable murecin lipoprotein signal peptide (Lpp-BRP) is not capable of inducing the release of cloacin DF13. To investigate the role of the stable BRP signal peptide in the release of cloacin DF13, the stable BRP signal peptide and the Lpp-BRP were expressed in trans in cells also producing cloacin DF13. Expression and release experiments indicate that the stable signal peptide can complement the Lpp-BRP in the release of cloacin DF 13.

Bacterial Proteins↗

PubMed1995

Optimization of bacteriocin-release-protein-induced protein release by Escherichia coli: extracellular production of the periplasmic molecular chaperone FaeE.

Expression of the pCloDF13-encoded bacteriocin-release protein (BRP) results in the release of periplasmic proteins into the culture medium. The BRP-mediated release of a periplasmic protein was investigated and optimized. As a periplasmic model protein, the 50-kDa dimeric E., coli fimbrial molecular chaperone FaeE was used. Plasmids were constructed for the simultaneous expression of the BRP and FaeE, controlled by independently inducible promoters. The efficiency of FaeE release increased when the BRP was targeted by the unstable murein lipoprotein signal peptide, instead of by its own stable signal, peptide. Furthermore, optimal efficacy of FaeE release was found when cells of E. coli strain C600 were used, which harboured one plasmid encoding both FaeE and BRP instead of two separate plasmids and which were cultured at 37 degrees C in broth supplemented with MgCl2. Maximal production levels of 21 mg FaeE/l culture were obtained.

Bacterial Proteins↗

PubMed1995

Expression of the pCloDF13 encoded bacteriocin release protein or its stable signal peptide causes early effects on protein biosynthesis and Mg2+ transport.

The effect of the pCloDF13 encoded bacteriocin release protein (BRP) on Escherichia coli cell lethality was studied. Induction of the BRP resulted in a strong inhibition of the incorporation of radioactive labeled amino acids and affected the transport of Mg2+ ions. Similar effects were obtained when the BRP stable signal peptide was expressed as a separate entity. Kinetic studies revealed that these effects occurred prior to quasi-lysis and release of cloacin DF13. The results indicated that the BRP induced cell lethality is caused by early effects on protein synthesis and Mg2+ transport, due to the accumulation of stable BRP signal peptides in the cytoplasmic membrane.

Amino Acids↗

PubMed1995

Bacteriocin release proteins: mode of action, structure, and biotechnological application.

The mechanism by which Gram-negative bacteria like Escherichia coli secrete bacteriocins into the culture medium is unique and quite different from the mechanism by which other proteins are translocated across the two bacterial membranes, namely through the known branches of the general secretory pathway. The release of bacteriocins requires the expression and activity of a so-called bacteriocin release protein and the presence of the detergent-resistant phospholipase A in the outer membrane. The bacteriocin release proteins are highly expressed small lipoproteins which are synthesized with a signal peptide that remains stable and which accumulates in the cytoplasmic membrane after cleavage. The combined action of these stable, accumulated signal peptides, the lipid-modified mature bacteriocin release proteins (BRPs) and phospholipase A cause the release of bacteriocins. The structure and mode of action of these BRPs as well as their application in the release of heterologous proteins by E. coli is described in this review.

Amino Acid Sequence↗

PubMed1994

Stability and function of the signal peptide of the pCloDF13-derived bacteriocin release protein.

The pCloDF13-derived bacteriocin release protein (BRP) is synthesized as a prelipoprotein with a signal peptide which remains stable after processing. This signal peptide accumulates in the cytoplasmic membrane and is, together with the mature BRP, required for efficient release of cloacin DF13. We investigated the structural requirements for stability of the BRP signal peptide by constructing hybrid signal peptides consisting of parts of the BRP and Lpp signal peptides. Signal peptide stability was investigated by pulse-labelling and pulse-chase experiments. To study the functioning of the BRP signal peptide, the hybrid constructs were tested for their ability to promote BRP-mediated cloacin DF13-release and their ability to affect the viability of the host cells. The results obtained suggest that the N-terminal part of the BRP signal peptide together with the C-terminal alanine residue are important for stability. When expressed as a separate entity, all mutant signal peptides that contain a part of the BRP signal peptide are capable of affecting cell viability. The results indicated a possible correlation between stability of the BRP signal peptide and cloacin DF13-release.

Alanine↗

PubMed1993

Escherichia coli SecB, SecA, and SecY proteins are required for expression and membrane insertion of the bacteriocin release protein, a small lipoprotein.

The SecB, SecA, and SecY dependency of a small outer membrane lipoprotein in Escherichia coli, the bacteriocin release protein (BRP), was studied. The detrimental effect of BRP expression on the culture turbidity (quasi-lysis) was strongly reduced in the sec mutants. Immunoblotting and radioactive labeling experiments showed that the expression, membrane insertion, and processing of the BRP precursor are dependent on SecB, SecA, and SecY. Labeling experiments with hybrid BRP gene constructs revealed that the mature part of the BRP precursor and not its stable signal sequence is important for its SecB dependency.

Adenosine Triphosphatases↗

PubMed1992

Lysozyme expression in Lactococcus lactis.

Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and lambda were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.

Bacteriophage lambda↗

PubMed1992

The stable BRP signal peptide causes lethality but is unable to provoke the translocation of cloacin DF13 across the cytoplasmic membrane of Escherichia coli.

The bacteriocin release protein (BRP) mediates the secretion of cloacin DF13. The BRP precursor is slowly processed to yield the mature BRP and its stable signal peptide which is also involved in cloacin DF13 secretion. The function of the stable BRP signal peptide was analysed by constructing two plasmids. First, the stable BRP signal peptide was fused to the murein lipoprotein and, second, a stop codon was introduced after the BRP signal sequence. Exchange of the unstable murein lipoprotein signal peptide for the stable BRP signal peptide resulted in an accumulation of precursors of the hybrid murein lipoprotein. This indicated that the BRP signal peptide, as part of this hybrid precursor, is responsible for the slow processing. The stable BRP signal peptide itself was not able to direct the transfer of cloacin DF13 into the periplasmic space or into the culture medium. Over-expression of the BRP signal peptide was lethal and caused 'lysis'. Subcellular fractionation experiments revealed that the BRP signal peptide is located exclusively in the cytoplasmic membrane whereas the mature BRP, targeted by either the stable BRP signal peptide or the unstable Lpp signal peptide, is located in both the cytoplasmic and outer membrane. These results are in agreement with the hypothesis that the stable signal peptide and the mature BRP together are required for the passage of cloacin DF13 across the cell envelope.

Amino Acid Sequence↗

PubMed1989

Binding-protein-dependent alanine transport in Rhodobacter sphaeroides is regulated by the internal pH.

The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R. sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F. E. Nano, Ph.D. thesis, University of Illinois, Urbana, 1984). Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M. G. L. Elferink, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1986). The results presented in this paper offer an explanation for these findings. Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system. The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3). The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system. Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles. This indicates that the H+/lactose symport system is active without electron transfer. Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport. Uptake of TMG and AIB is regulated by the internal pH. The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB. At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8. In a previous study, we found that K+ plays an essential role in regulating the internal pH (T. Abee, K. J. Hellingwerf, and W. N. Konings, J. Bacteriol. 170:5647-5653, 1988). The dependence of solute transport in R. sphaeroides on both K+ and activity of an electron transfer chain can be explained by an effect of the internal pH, which subsequently influences the activities of the lactose-and binding-protein-dependent L-alanine transport system.

Alanine↗