Anton Schmitz

Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance.

G proteins are an important class of regulatory switches in all living systems. They are activated by guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP for GTP. This activity makes GEFs attractive targets for modulating disease-relevant G-protein-controlled signalling networks. GEF inhibitors are therefore of interest as tools for elucidating the function of these proteins and for therapeutic intervention; however, only one small molecule GEF inhibitor, brefeldin A (BFA), is currently available. Here we used an aptamer displacement screen to identify SecinH3, a small molecule antagonist of cytohesins. The cytohesins are a class of BFA-resistant small GEFs for ADP-ribosylation factors (ARFs), which regulate cytoskeletal organization, integrin activation or integrin signalling. The application of SecinH3 in human liver cells showed that insulin-receptor-complex-associated cytohesins are required for insulin signalling. SecinH3-treated mice show increased expression of gluconeogenic genes, reduced expression of glycolytic, fatty acid and ketone body metabolism genes in the liver, reduced liver glycogen stores, and a compensatory increase in plasma insulin. Thus, cytohesin inhibition results in hepatic insulin resistance. Because insulin resistance is among the earliest pathological changes in type 2 diabetes, our results show the potential of chemical biology for dissecting the molecular pathogenesis of this disease.

The biological role of the Alzheimer amyloid precursor protein in epithelial cells

Abstract. Proteolytic processing of the Alzheimer amyloid precursor protein (APP) results in the generation of at least two distinct classes of biologically relevant peptides: (1) the amyloid beta peptides which are believed to be involved in the pathogenesis of Alzheimers disease and (2) the soluble N-terminal ectodomain (sAPP) which exhibits a protective but as yet ill-defined effect on neurons and epithelial cells. In this report we present an overview on the functions of sAPP as an epithelial growth factor. This function involves specific binding of sAPP to membrane rafts and results in signal transduction and various physiological effects in epithelial cells as different as keratinocytes and thyrocytes. At nanomolar concentrations sAPP induces a two to fourfold increase in the rate of cell proliferation and cell migration. Specific inhibition of APP expression by antisense techniques results in decreased sAPP release and in reduced proliferative and motogenic activities. Proliferation and migration are known to be part of complex processes such as wound healing which, therefore, might be facilitated by the growth factor function of sAPP.

Endoplasmic reticulum-localized amyloid beta-peptide is degraded in the cytosol by two distinct degradation pathways.

The paradigm of endoplasmic reticulum (ER)‐associated degradation (ERAD) holds that misfolded secretory and membrane proteins are translocated back to the cytosol and degraded by the proteasome in a coupled process. Analyzing the degradation of ER‐localized amyloid β‐peptide (Aβ), we found a divergence from this general model. Cell‐free reconstitution of the export in biosynthetically loaded ER‐derived brain microsomes showed that the export was mediated by the Sec61p complex and required a cytosolic factor but was independent of ATP. In contrast to the ERAD substrates known so far, the exported Aβ was degraded by both, a proteasome‐dependent and a proteasome‐independent pathway. RNA interference experiments in Aβ‐transfected cells identified the protease of the proteasome‐independent pathway as insulin‐degrading enzyme (IDE). The IDE‐mediated clearance mechanism for ER‐localized Aβ represents an as yet unknown type of ERAD which is not entirely dependent on the proteasome.

Cytosolic and nuclear aggregation of the amyloid β-peptide following its expression in the endoplasmic reticulum

Abstract. Misfolded secretory and membrane proteins are known to be exported from the endoplasmic reticulum (ER) to the cytosol where they are degraded by proteasomes. When the amount of exported misfolded proteins exceeds the capacity of this degradation mechanism the proteins accumulate in the form of pericentriolar aggregates called aggresomes. Here, we show that the amyloid β-peptide (Aβ) forms cytosolic aggregates after its export from the ER. These aggregates share several constituents with aggresomes. However, Aβ aggregates are distinct from aggresomes in that they do not accumulate around the centrosome but are distributed randomly around the nucleus. In addition to these cytosolic aggregates, Aβ forms intranuclear aggregates which have as yet not been found for proteins exported from the ER. These findings show that proteins exported from the ER to the cytosol which escape degradation by the proteasome are not necessarily incorporated into aggresomes. We conclude that several distinct aggregation pathways may exist for proteins exported from the ER to the cytosol.

Endoplasmic reticulum-associated degradation: exceptions to the rule

Quality control mechanisms in the endoplasmic reticulum (ER) ensure that misfolded proteins are recognized and targeted for degradation. According to the current view of ER-associated degradation (ERAD), the degradation does not occur in the ER itself but requires the retrotranslocation of the proteins to the cytosol where they are degraded by proteasomes. Although this model appears to be valid for many different proteins a number of exceptions from this rule suggest that additional proteasome-independent ERAD pathways may exist. In this review, we will summarize what is known about these alternative ERAD pathways.

BiP-dependent export of cholera toxin from endoplasmic reticulum-derived microsomes

Cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) from where it is translocated to the cytosol in a process depending on ATP and luminal ER proteins. To test whether the molecular chaperone BiP (heavy chain binding protein), which is an ER‐luminal ATPase, was one of the required proteins the export of CT was analyzed using ER‐derived CT‐loaded microsomes. The resubstitution of extracted export‐incompetent microsomes with purified BiP was sufficient to restore the export of CT. As BiP protected CT from aggregation it is proposed that BiP maintains CT in a soluble, export‐competent state.

Biological roles of APP in the epidermis.

The amyloid precursor protein (APP) was initially detected in cells of the central nervous system where it is considered to be involved in the pathogenesis of Alzheimers disease. However, APP is also found in peripheral organs with exceptionally strong expression in the mammalian epidermis where it fulfils a variety of distinct biological roles. Full length APP appears to facilitate keratinocyte adhesion due to its ability to interact with the extracellular matrix. The C-terminus of APP also serves as adapter protein for binding the motor protein kinesin thereby mediating the centripetal transport of melanosomes in epidermal melanocytes. By the action of alpha-secretase sAPPalpha, the soluble N-terminal portion of APP, is released. sAPPalpha has been shown to be a potent epidermal growth factor thus stimulating proliferation and migration of keratinocytes as well as the exocytic release of melanin by melanocytes. The release of sAPPalpha can be almost completely blocked by inhibiting alpha-secretase with hydroxamic acid-based zinc metalloproteinase inhibitors. In hyperproliferative keratinocytes from psoriatic skin this inhibition results in normalized growth.

Covalent cross-linking of secreted bovine thyroglobulin by transglutaminase

Extracellular storage of thyroglobulin (TG) is a prerequisite for maintaining constant levels of thyroid hormones in vertebrates. Storage of TG within the follicle lumen is achieved by compactation and by the formation of covalent cross‐links between TG molecules. In bovine thyroids, ~75% of the cross‐links are other than disulfide bonds (J. Cell Biol. 180, 1071–1081). We have now shown that polymeric TG contains a large number of Nε(γ‐glutamyl)lysine cross‐links and that only traces of these can be found in the soluble form of TG. Because such isopeptide bridges are generated usually by the action of a transglutaminase, it is reasonable to propose that the covalent polymerization of TG in the globules is under the control of this enzyme. Soluble TG was shown to be a substrate for transglutaminase in vitro; moreover, the presence of transglutaminase was demonstrated by immunofluo‐rescence and by immunoblotting in freshly isolated bovine thyroid globules. With immunoelectron microscopy, transglutaminase was detected in the cytoplasm of thyrocytes, but not in compartments of the secretory pathway. Only one messenger RNA for transglutaminase was found by Northern blotting. Sequencing of the cloned gene failed to reveal a secretory signal, which supports the notion that the thyroid transglutaminase is the cytosolic type. Apparently, the enzyme reaches the lumen of the follicle by an as yet unknown pathway to catalyze the covalent cross‐linking of thyroid globules in this extracellular compartment.—Saber‐Lichtenberg, Y., Brix, K., Schmitz, A., Heuser, J. E., Wilson, J. H., Lorand, L., and Herzog, V. Covalent cross‐linking of secreted bovine thyroglobulin by transglutaminase FASEB J. 14, 1005–1014 (2000)

Targeting multifunctional proteins by virtual screening: structurally diverse cytohesin inhibitors with differentiated biological functions

Virtual screening (VS) of chemical libraries formatted in silico provides an alternative to experimental high-throughput screening (HTS) for the identification of small molecule modulators of protein function. We have tailored a VS approach combining fingerprint similarity searching and support vector machine modeling toward the identification of small molecular probes for the study of cytohesins, a family of cytoplasmic regulator proteins with multiple cellular functions. A total of 40 new structurally diverse inhibitors were identified, and 26 of these compounds were more active than the primary VS template, a single known inhibitory chemotype, in at least one of three different assays (guanine nucleotide exchange, Drosophila insulin signaling, and human leukocyte cell adhesion). Moreover, these inhibitors displayed differential inhibitory profiles. Our findings demonstrate that, at least for the cytohesins, computational extrapolation from known active compounds was capable of identifying small molecular probes with highly diversified functional profiles.

Misfolded BiP is degraded by a proteasome-independent endoplasmic-reticulum-associated degradation pathway

Misfolded proteins are removed from the ER (endoplasmic reticulum) by retrotranslocation to the cytosol and degradation by the ubiquitin-proteasome system in a process designated ERAD (ER-associated degradation). Analysing the turnover of a misfolded form of the ER-resident chaperone BiP (heavy-chain binding protein) (BiPDeltaA), we found that the degradation of BiPDeltaA did not follow this general ERAD pathway. In transfected cells, BiPDeltaA was degraded, although proteasome-dependent ERAD was inactivated either by proteasome inhibitors or by ATP depletion. In semi-permeabilized cells, which did not support the degradation of the proteasomal substrate alpha1-antitrypsin, the degradation of BiPDeltaA was still functional, excluding the Golgi apparatus or lysosomes as the degradative compartment. The degradation of BiPDeltaA was recapitulated in biosynthetically loaded brain microsomes and in an extract of luminal ER proteins. In contrast with proteasome-dependent ERAD, degradation fragments were detectable inside the microsomes and in the extract, and the degradation was prevented by a serine protease inhibitor. These results show that the degradation of BiPDeltaA was initiated in the ER lumen by a serine protease, and support the view that proteasome-independent ERAD pathways exist.