Volume 382, Issue 7

Identification and characterization of proteinprotein interactions between the host cell and parasites both enhance our understanding of basic cell biology and provide insights into central processes of parasite life cycles. Research on HIV-1 has broadened our knowledge of the various molecular events involved. However, our understanding of how this virus interacts with the host cell at the level of proteinprotein interaction is still limited. Through these interactions the virus is able to recruit certain cellular metabolic pathways for its replication. Here we summarize our current knowledge of proteinprotein interactions between HIV-1 and host cell factors during viral replication.

A putative operon of the ribosomal proteins S20 and Thx has been determined in a 1.4 kb sequenced region of T. thermophilus genomic DNA. Both genes have a promoter sequence 29 nt upstream of ORF1, possess their own ShineDalgarno motifs (GGAG) and are separated by only 9 nucleotides, a feature characteristic of the compact Thermus thermophilus genome. This is a novel arrangement, since Thx is unique to the Thermus bacteria and in all other prokaryotes the S20 gene is monocistronic. Our results, in conjunction with the recent finding that Thx is located on the top of the head of the 30S subunit in a cavity between multiple RNA elements stabilizing them with its positive charge, corroborate the observation that thermophilic ribosomes require constituents with special features for their stabilization at high temperatures.

An RNA sequence showing high stability with respect to digestion by ribonuclease T1 (RNase T1) was isolated by in vitro selection from an RNA library. Although ribonuclease T1 cleaves singlestranded RNA specifically after guanosine residues, secondary structure calculations predict several guanosines in singlestranded areas. Two of these guanosines are part of a GGCAtetraloop, a recurring structure element in the secondary structure predictions. Molecular dynamics simulations of the conformation space of the nucleotides involved in this tetraloop show on the one hand that the nucleic acid backbone of the guanosines cannot realise the conformation required for cleavage by RNase T1. On the other hand, it could be shown that an RNA molecule not forced into a tetraloop occupies this conformation several times in the course of the simulation. The simulations confirm the GGCAtetraloop as an RNasestable secondary structure element. Our results show that, besides the known prerequisite of a singlestranded RNA, RNase T1 has additional demands on the substrate conformation.

Recently, γglutamyl transpeptidase, which initiates cleavage of extracellular glutathione, has been shown to promote oxidative damage to cells. Here we examined a murine disease model of glomerulosclerosis, involving loss of the Mpv17 gene coding for a peroxisomal protein. In Mpv17[/] cells, enzyme activity and mRNA expression (examined by quantitative RTPCR) of membranebound γglutamyl transpeptidase were increased, while plasma glutathione peroxidase and superoxide dismutase levels were lowered. Superoxide anion production in these cells was increased as documented by electron spin resonance spectroscopy. In the presence of Mn(III)tetrakis(4- benzoic acid)porphyrin, the activities of γglutamyl transpeptidase and plasma glutathione peroxidase were unchanged, suggesting a relationship between enzyme expression and the amount of reactive oxygen species. Inhibition of γglutamyl transpeptidase by acivicin reverted the lowered plasma glutathione peroxidase and superoxide dismutase activities, indicating reciprocal control of gene expression for these enzymes.

It has been previously suggested that juvenile hormone binding protein(s) (JHBP) belongs to a new class of proteins. In the search for other protein(s) that may contain structural motifs similar to those found in JHBP, hemolymph from Galleria mellonella (Lepidoptera) was chromatographed over a Sephadex G 200 column and resulting fractions were subjected to SDSPAGE, transferred onto nitrocellulose membrane and scanned with a monoclonal antibody, mAb 104, against hemolymph JHBP. Two proteins yielded a positive reaction with mAb 104, one corresponding to JHBP and the second corresponding to a transferrin, as judged from Nterminal amino acid sequencing (KPNYKIXVPQKFLKEXEQMLEVXT) and Ferene S staining. Transferrin was purified to about 80% homogeneity using a twostep procedure including Sephadex G-200 gel filtration and HPLC MonoQ column chromatography. Panning of a random peptide display library and analysis with immobilized synthetic peptides were applied for finding a common epitope present in JHBP and the transferrin molecule. The postulated epitope motif recognized by mAb 104 in the JHBP sequence is RDTKAVN, and is localized at position 82 88.

Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the cotranslational transfer of GlcNAc[2]Man[9]Glc[3] from Dol PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ERtargeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man[9] mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the Cterminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48?K) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ERresidency on OST48. The doublelysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48?K-3, when coexpressed in COS-1 cells together with myctagged ribophorin I, was quantitatively retained in the ER. By contrast, coexpression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMO?K-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the Cterminus of the Man[9]mannosidase luminal domain. Thus ERlocalisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the doublelysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in recapture of molecules which have escaped from the ER.

Irreversible binding of Teven bacteriophages to Escherichia coli is mediated by the short tail fibres, which serve as inextensible stays during DNA injection. Short tail fibres are exceptionally stable elongated trimers of gene product 12 (gp12), a 56 kDa protein. The Nterminal region of gp12 is important for phage attachment, the central region forms a long shaft, while a Cterminal globular region is implicated in binding to the bacterial lipopolysaccharide core. When gp12 was treated with stoichiometric amounts of trypsin or chymotrypsin at 37 C, an Nterminally shortened fragment of 52 kDa resulted. If the protein was incubated at 56 C before trypsin treatment at 37 C, we obtained a stable trimeric fragment of 3 33 kDa lacking residues from both the N and Ctermini. Apparently, the protein unfolds partially at 56 C, thereby exposing proteasesensitive sites in the Cterminal region and extra sites in the Nterminal region. Welldiffracting crystals of this fragment could be grown. Our results indicate that gp12 carries a stable central region, consisting of the Cterminal part of the shaft and the attached Nterminal half of the globular region. Implications for structure determination of the gp12 protein and its folding are discussed.

Insulininduced cell swelling was recently suggested to reflect an independent signal for metabolic insulin effects such as inhibition of hepatic proteolysis, which is transmitted at the level of autophagosome formation via p38 activation [Häussinger et al., Gastroenterology 116 (1999), 921 935]. Here, the role of insulininduced cell swelling in the overall context of insulin signalling towards proteolysis inhibition was studied in perfused rat liver. Loop diuretics and hyperosmolarity, which impair insulinstimulated cell swelling, strongly blunt Erk-2 and p38 activation as well as proteolysis inhibition by insulin, but are without effect on insulininduced tyrosine phosphorylation of IRβ and IRS-1. Inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) also block insulininduced cell swelling, MAP kinase activation and proteolysis inhibition, but the antiproteolytic response to hypoosmolarity remains unaffected. We suggest that PI3-kinasemediated cell swelling induced by insulin is required to amplify the insulin signal to MAP kinases and thus proteolysis regulation. The perturbation of insulininduced cell swelling may be of pathophysiological relevance for the development of insulin resistance in clinical situations associated with hyperosmotic dehydration and loop diuretic treatment.

The electronically excited molecular oxygen (singlet oxygen, [1]O[2]) can be detrimental to cells in several ways, although recent reports indicate that it may play a role as an intercellular signal in eukaryotes. Here we present evidence that [1]O[2], generated by thermodissociation of disodium 3,3(1,4-naphthylidene) diproprionate endoperoxide, activates transcription of genes of the soxRS regulon, and that this induction is paralleled by induction of a soxS::lacZ operon fusion. The inductions were dependent on a functional soxR gene. These data imply that protective responses, such as induction of the soxRS regulon, may be triggered by diverse environmental oxidative stresses, and that [1]O[2] may also function as a signal molecule in prokaryotes.

The zinc finger protein early growth response 1 (Egr 1) is a transcriptional activator involved in the regulation of growth and differentiation. We show here that a constitutive active mutant of mitogenactivated kinase kinase-1 (MAPKK-1) strongly stimulates the activity of the Egr-1 promoter, thus explaining the effects of mitogens upon Egr-1 mRNA and protein levels. Moreover, we show that a constitutive active MAPKK-1 leads to an increase in the biological activity of Egr-1 to activate transcription. We conclude that the signaling pathway involving mitogenactivated protein kinase/extracellular signalregulated protein kinase has a dual impact on the biology of Egr-1 by controlling the transcription of the Egr-1 gene and the transcriptional activity of the Egr-1 protein.

The transcription of the fatty acid synthase (FAS) gene is regulated by the sterol status of the cell via cleavage of the sterol regulatory elementbinding protein (SREBP). When human HepG2 hepatoma cells were cotransfected with an expression plasmid for mature SREBP-1a together with FAS promoter/reporter constructs significant increases in reporter activity were observed. Deletion analysis of the FAS promoter between 151 and 52 relative to the transcription start site pinpoint two ciselements important in sterol regulation of the FAS gene. One element, FIRE3, between 71 and 52 can bind in vitro translated and transcribed SREBP-1a whereas the other element, the inverted CCAAT element ICE( 97/ 92), binds the trimeric transcription factor NFY/CBF as shown with rat liver extract and reconstituted, recombinant NFY. The results clearly show that the coactivator for SREBP-1a in this cell line is NFY. This finding was confirmed by using a dominant negative form of NFYA, NFYAm29, which interferes with the effect of ectopically expressed SREBP-1a on FAS reporter activity.

Complement factor I (FI) is a regulatory serine protease of the complement system which cleaves three peptide bonds in the αchain of C3b and two bonds in the αchain of C4b and thus prevents the assembly of the C3 and C5 convertases. We have investigated the proinflammatory cytokines IL-6, IL-1β, TNFα and IFNγ for their potential role in the regulation of FI expression. Of the investigated cytokines, only IL-6 increased the FIspecific RTPCR signal in isolated hepatocytes, in the two rat hepatomaderived cell lines FAO and H4IIE or in HUVECs. Quantitative competitive RTPCR showed an IL-6 induced upregulation of FIspecific mRNA by about tenfold. These data are in accord with Northern blot analyses in which the FImRNA was upregulated by IL-6 between five and sevenfold. IL-6, but not IL-1β, TNFα or IFNγ also increased FIprotein levels in cell culture supernatants by about fivefold as determined by a semiquantitative immunoblot using a novel monoclonal antibody specific for rat FI.

Infection by Staphylococcus epidermidis, an opportunistic pathogen, has become a major problem due to the increased use of implanted medical devices and the growing number of patients who are therapeutically or infectiously immunosuppressed. These infections appear to proceed via modulation of the coagulation and complement systems. In this communication we describe the purification and characterization of a novel extracellular proteinase from an oral strain of S. epidermidis that can degrade fibrinogen, complement protein C5, and several other proteins. This proteinase has a strong preference for cleavage after glutamic acid residues, but not after aspartic acid. The S. epidermidis enzyme may be a multifunctional protein which not only provides this organism with both the ability to evade the complement defense system and to dysregulate the coagulation cascade, but also supplies nutrients for its growth through the degradation of Glurich proteins.