Siegfried Ansorge

The N-terminal Structure of HIV-1 Tat Is Required for Suppression of CD26-dependent T Cell Growth

Evidence exists that the human immunodeficiency virus-1 (HIV-1) transactivator Tat occurs extracellularly and is involved in the immunosuppression of non-HIV-1-infected T cells of acquired immunodeficiency syndrome (AIDS) patients. The mechanism of this immunosuppressive activity of Tat has been controversially discussed. Interestingly, Tat binds to the T cell activation marker CD26, which has been shown to play a key role in the regulation of growth of lymphocytes and to inhibit its dipeptidyl peptidase IV (DP IV) activity. Here we show that the N-terminal nonapeptide MDPVDPNIE of Tat is a competitive inhibitor of DP IV and suppresses DNA synthesis of tetanus toxoid-stimulated peripheral blood mononuclear cells. Amino acid exchanges at positions 5 and 6 strongly weaken these effects.1H nuclear magnetic resonance and molecular dynamics simulations of Tat(1–9), I5-Tat(1–9), and L6-Tat(1–9) suggest a similar backbone conformation for Tat(1–9) and L6-Tat(1–9). The solution conformation of I5-Tat(1–9) considerably differs from the other two. However, Tat(1–9) fits into our previously proposed active site model of DP IV in contrast to I5-Tat(1–9) and L6-Tat(1–9). Conformational alterations with regard to the parent peptide and spatial hindrances between these both compounds and DP IV can explain the loss of inhibitory activity. Our data suggest that the N-terminal residues of HIV-1 Tat do interact directly with the active site of DP IV and that DP IV does mediate Tat’s immunosuppressive effects.

Down-regulation of T Cell Activation following Inhibition of Dipeptidyl Peptidase IV/CD26 by the N-terminal Part of the Thromboxane A2 Receptor

Using synthetic inhibitors, it has been shown that the ectopeptidase dipeptidyl peptidase IV (DP IV) (CD26) plays an important role in the activation and proliferation of T lymphocytes. The human immunodeficiency virus-1 Tat protein, as well as the N-terminal nonapeptide Tat(1–9) and other peptides containing the N-terminal sequence XXP, also inhibit DP IV and therefore T cell activation. Studying the effect of amino acid exchanges in the N-terminal three positions of the Tat(1–9) sequence, we found that tryptophan in position 2 strongly improves DP IV inhibition. NMR spectroscopy and molecular modeling show that the effect of Trp2-Tat(1–9) could not be explained by significant alterations in the backbone structure and suggest that tryptophan enters favorable interactions with DP IV. Data base searches revealed the thromboxane A2 receptor (TXA2-R) as a membrane protein extracellularly exposing N-terminal MWP. TXA2-R is expressed within the immune system on antigen-presenting cells, namely monocytes. The N-terminal nonapeptide of TXA2-R, TXA2-R(1–9), inhibits DP IV and DNA synthesis and IL-2 production of tetanus toxoid-stimulated peripheral blood mononuclear cells. Moreover, TXA2-R(1–9) induces the production of the immunosuppressive cytokine transforming growth factor-β1. These data suggest that the N-terminal part of TXA2-R is an endogenous inhibitory ligand of DP IV and may modulate T cell activation via DP IV/CD26 inhibition.

The identity of the insulin degrading thiol-protein disulfide oxidoreductase (glutathione-insulin transhydrogenase) with the sulfhydryl-disulfide interchange enzyme.

In previous studies [1-3] we reported on a glutathione-insulin transhydrogenase from microsomes of rat liver. The substrate specificity of this enzyme is not restricted to insulin and glutathione. We were able to show that this enzyme catalyzes also the reduction of a number of other disulfide-containing proteins, e.g. trypsin, chymotrypsin, chymotrypsinogen, pepsin, bovine serum albumin, r-globulin, oxytocin, lysozyme, ribonuclease, randomly-oxidized ribonuclease, and the intermediate of proinsulin [4,5]. Because of the broad specificity we prefer the term thiol-protein disulfide oxidoreductase (TPO) instead of glutathione-insulin transhydrogenase (GIT) for this enzyme system (Katzen et al. [6]). The specificity of action of the TPO on proteinbound disulfide groups as well as its intracellular localization suggested that this enzyme might be identical with or closely related to the sulfhydryl-disulfide interchange enzyme (ribonuclease-reactivating enzyme) discovered by Anfinsen et al. [7] and also by Venetianer and Straub [8]. Indications supporting this assumption were given by Katzen and Tietze [6], Givol et al. [9] and Narahara [10], but convincing evidence as to our knowledge was lacking until now. The sulfhydryl-disulfide interchange enzyme has been shown to be present in several organs and was purified from bovine liver microsomes [7, 8, 11-13]. The present report gives conclusive evidence on the identity of the microsomal TPO and the sulfhydryl-disulfide interchange enzyme. Preliminary results concerning this problem have been published [3-5].

Transcellular Proteolysis Demonstrated by Novel Cell Surface-associated Substrates of Dipeptidyl Peptidase IV (CD26)

Proteolytic enzymes contribute to the regulation of cellular functions such as cell proliferation and death, cytokine production, and matrix remodeling. Dipeptidyl peptidase IV (DP IV) catalyzes the cleavage of several cytokines and thereby contributes to the regulation of cytokine production and the proliferation of immune cells. Here we show for the first time that cell surface-bound DP IV catalyzes the cleavage of specific substrates that are associated with the cellular surface of neighboring cells. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize dipeptidyl peptidase IV (DP IV/CD26) substrates Gly(Ala)-Pro-R110-R, thus facilitating a stable binding of the fluorescent moiety on the cell surface. The fixation resulted from the interaction with the reactive anchor rhodamine and allowed the quantification of cellular DP IV activity on single cells. The reactivity, length, and hydrophobicity of rhodamine was characterized as the decisive factor that facilitated the determination of cellular DP IV activity. Using fluorescence microscopy, it was possible to differentiate between different DP IV activities. The hydrolysis of cell-bound substrates Xaa-Pro-R110-R by DP IV of neighboring cells and by soluble DP IV was shown using flow cytometry. These data demonstrate that ectopeptidases such as DP IV may be involved in communication between blood cells via proteolysis of cell-associated substrates.

Präparative gewinnung hochgereinigter lysosomenenzyme aus rattenlebern

By means of a relatively simple procedure lysosomal enzymes from rat livers with 20% yield and a 50‐fold enrichment were prepared. Density gradient centrifugation or tissue‐preloading with triton VR 1339 is not necessary. Up to several liters of 50% liver homogenate may be used. Intact lysosomes are not obtained, because the crucial step in the tissue fractionation scheme is the selective release of lysosomal enzymes from a crude lysosome pellet by water extraction. The presence of neutral endopeptidases with high specific activities in lysosomes (estimated using azocasein and other proteins as substrates) is demonstrated.

Intracellular Protein Turnover

In the first part of this short review we shall summarize some of the main characteristics of intracellular protein turnover, and in the second part we shall go on to describe some of our work on the molecular mechanisms responsible for the characteristics of this process in rat liver. But first we must define the processes that we are talking about.

The activation-dependent induction of APN-(CD13) in T-cells is controlled at different levels of gene expression

Recently, it was shown that aminopeptidase N (E.C. 3.4.11.2, CD13) is up‐regulated during mitogenic stimulation of peripheral T‐cells. In this study, we demonstrate that the half‐life of APN mRNA was considerably prolonged in these cells leading to a 2.7‐fold increase of APN transcript level. The apparent half‐life time of the APN transcript was investigated by the RNA synthesis inhibitor‐chase method using actinomycin D. The steady‐state APN mRNA levels was determined by a competitive RT‐PCR. The half‐lives estimated in resting T‐cells, natural killer cells and permanently growing tumour cells varied between 3.5 and 6 h. Finally, nuclear run‐on assays revealed that the APN gene expression of stimulated T‐cells is controlled by increased promoter activity as well. These studies suggest a control of APN gene expression at the post‐transcriptional level in addition to promoter‐mediated regulation.

PROTEIN CATABOLISM IN RAT LIVER CELLS

Publisher Summary This chapter discusses protein catabolism in rat liver cells. The use of in vivo short- and long-lived proteins as substrates in vitro allows proving whether the conditions chosen in vitro might be a sufficient simulation of conditions in living cells, and also whether the proteinase used is important for the selective breakdown of short-lived substrate proteins. The protein degradation occuring in vitro in the microsomal fraction must not sufficiently simulate the in vivo process, because a selective autolytic and lysosomal degradation of the in vivo long-lived membrane proteins. Investigations of the primary steps of intracellular protein catabolism must include all organelles, especially the organelles in the microsomal fraction, the energy requirement, the possible role of microtubules and the distinction between basal and enhanced intracellular proteolysis.

The age dependence of intracellular proteolysis: Changes of the substrate proteins

Liver cytosol proteins of young (4--6 months) and old (18--27 months) rats were degraded in vitro by papain, pronase, trypsin, pepsin, cathepsin D from rat liver and a soluble lysosomal enzyme mixture from rat liver. We could demonstrate the capability of the latter enzyme mixture to degrade proteolytically the cytosol proteins of young animals about 20% faster than those of the older animal group. Digesting radioactive labelled young cytosol in the presence of unlabelled old cytosol the possibility could be excluded, that this effect was due to an inhibitor of macromolecular size present in the old cytosol.

Immunohistochemischer Nachweis der Thiol: Proteindisulfid-Oxidoreductase (TPO) im Pankreas und in isolierten Langerhansschen Inseln. Eine light-, fluoreszenz- und elektronenmikroskopische Studie

Zusammenfassung Die Thiol: Proteindisulfid Oxidoreductase (E. C. 1.8.4.2) kann die Thiol-Disulfid-Austausch-reaktionen bei den simultan verlaufenden Vorgangen der Proteinbiosynthese und des zellularen Proteinabbaus katalysieren. Mit Hilfe der immunohistochemischen Technik ist es gelungen, dieses Enzym verstarkt in den Langerhansschen Inseln und im geringeren Grade in den Acinuszellen des Pankreas nachzuweisen. Elektronenmikroskopische Untersuchungen an isolierten Langerhansschen Inseln zeigen eine Lokalisation des ubiquitar vorkommenden Enzyms an der auseren Kernmembran, den Membranen des endoplasmatischen Reticulums und der B-Zell-Granula als auch am Plasmalemm. Die genannten Strukturen stehen im Zusammenhang mit der Insulinbiosynthese und der Insulinabgabe. Es sind gute Korrelationen zwischen morphologischen und biochemischen Befunden erzielt worden. In den Acinuszellen finden wir geringe Reaktionsniederschlage an der auseren Kernmembran, den Membranen des endoplasmatischen Reticulums und am Plasmalemm.