Volker Herzog

Membrane and raft association of reggie-1/flotillin-2 : role of myristoylation, palmitoylation and oligomerization and induction of filopodia by overexpression

The reggie protein family consists of two proteins, reggie-1 and -2, also called flotillins, which are highly ubiquitous and evolutionarily conserved. Both reggies have been shown to be associated with membrane rafts and are involved in various cellular processes such as T-cell activation, phagocytosis and insulin signalling. However, the exact molecular function of these proteins remains to be determined. In addition, the mechanism of membrane association of reggie-1, which does not contain any transmembrane domain, is not known. In this study, we have produced a fusion protein of reggie-1 with enhanced green fluorescent protein and generated targeted substitutions for the inactivation of putative palmitoylation and myristoylation sites. We were able to show that reggie-1 is myristoylated and multiply palmitoylated and that lipid modifications are necessary for membrane association of reggie-1. Overexpression of reggie-1 resulted in the induction of numerous filopodia-like protrusions in various cell lines, suggesting a role for reggie-1 as a signalling protein in actin-dependent processes.

Thyroid functions of mouse cathepsins B, K, and L

Thyroid function depends on processing of the prohormone thyroglobulin by sequential proteolytic events. From in vitro analysis it is known that cysteine proteinases mediate proteolytic processing of thyroglobulin. Here, we have analyzed mice with deficiencies in cathepsins B, K, L, B and K, or K and L in order to investigate which of the cysteine proteinases is most important for proteolytic processing of thyroglobulin in vivo. Immunolabeling demonstrated a rearrangement of the endocytic system and a redistribution of extracellularly located enzymes in thyroids of cathepsin-deficient mice. Cathepsin L was upregulated in thyroids of cathepsin K(-/-) or B(-/-)/K(-/-) mice, suggesting a compensation of cathepsin L for cathepsin K deficiency. Impaired proteolysis resulted in the persistence of thyroglobulin in the thyroids of mice with deficiencies in cathepsin B or L. The typical multilayered appearance of extracellularly stored thyroglobulin was retained in cathepsin K(-/-) mice only. These results suggest that cathepsins B and L are involved in the solubilization of thyroglobulin from its covalently cross-linked storage form. Cathepsin K(-/-)/L(-/-) mice had significantly reduced levels of free thyroxine, indicating that utilization of luminal thyroglobulin for thyroxine liberation is mediated by a combinatory action of cathepsins K and L.

p-ANCAs in autoimmune liver disorders recognise human b-tubulin isotype 5 and cross-react with microbial protein FtsZ

Objective Autoimmune hepatitis and primary sclerosing cholangitis are chronic inflammatory disorders of unknown aetiology, frequently associated with the presence of perinuclear antineutrophil cytoplasmic antibodies (p-ANCAs) directed against an unknown antigen of myeloid cells. Methods and Results Here, it is reported that p-ANCAs in autoimmune liver disorders react with β-tubulin isotype 5 (TBB-5) as autoantigen as well as with its evolutionary bacterial precursor protein FtsZ. Both proteins were confirmed as antigens of p-ANCAs in autoimmune liver disorders by demonstrating reactivity of ANCA-positive sera with recombinant TBB-5 (72–88%) and FtsZ (64–82%) on immunoblots and antigen-specific abrogation of ANCA immunofluorescence when sera had been preabsorbed with tubulin and FtsZ. Using sera from interleukin 10-deficient mice (Il10–/–), an animal model of inflammatory bowel disease, it was also demonstrated that antibodies against TBB-5 are generated in response to intestinal microorganisms. However, unlike autoimmune liver disorders, human antibodies to FtsZ in the absence of TBB-5 antibodies were also a frequent finding in non-autoimmune liver diseases (up to 95%). Reactivity to TBB-5 without the presence of FtsZ antibodies was found in very few cases (<1%) in autoimmune liver disorders. Conclusions Thus, p-ANCAs in autoimmune liver diseases are directed against human TBB-5 cross-reacting with the bacterial protein FtsZ, probably reflecting an abnormal immune response to intestinal microorganisms in susceptible, possibly genetically predisposed individuals.

Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1

To address the functions of Rac1 in keratinocytes of the basal epidermal layer and in the outer root sheath of hair follicles, we generated transgenic mice expressing a dominant inhibitory mutant of Rac, N17Rac1, under the control of the keratin 14 promoter. These mice do not exhibit an overt skin phenotype but show protracted skin wound re-epithelialization. Investigation into the underlying mechanisms revealed that in vivo both proliferation of wound-edge keratinocytes and centripetal migration of the neo-epidermis were impaired. Similar results were obtained in mice with an epidermis-specific deletion of Rac1. Primary epidermal keratinocytes that expressed the N17Rac1 transgene were less proliferative than control cells and showed reduced ERK1/2 phosphorylation upon growth factor stimulation. Adhesion, spreading, random migration and closure of scratch wounds in vitro were significantly inhibited on collagen I and, to a lesser extent, on fibronectin. Stroboscopic analysis of cell dynamics (SACED) of N17Rac1 transgenic and control keratinocytes identified decreased lamella-protrusion persistence in connection with increased ruffle frequency as a probable mechanism for the observed impairment of keratinocyte adhesion and migration. We conclude that Rac1 is functionally required for normal epidermal wound healing and, in this context, exerts a dual function – namely the regulation of keratinocyte proliferation and migration.

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.

Trafficking of lysosomal cathepsin B—green fluorescent protein to the surface of thyroid epithelial cells involves the endosomal/lysosomal compartment

Cathepsin B, a lysosomal cysteine proteinase, is involved in limited proteolysis of thyroglobulin with thyroxine liberation at the apical surface of thyroid epithelial cells. To analyze the trafficking of lysosomal enzymes to extracellular locations of thyroid epithelial cells, we have expressed a chimeric protein consisting of rat cathepsin B and green fluorescent protein. Heterologous expression in CHO cells validated the integrity of the structural motifs of the chimeric protein for targeting to endocytic compartments. Homologous expression, colocalization and transport experiments with rat thyroid epithelial cell lines FRT or FRTL-5 demonstrated the correct sorting of the chimeric protein into the lumen of the endoplasmic reticulum, and its subsequent transport via the Golgi apparatus and the trans-Golgi network to endosomes and lysosomes. In addition, the chimeras were secreted as active enzymes from FRTL-5 cells in a thyroid-stimulating-hormone-dependent manner. Immunoprecipitation experiments after pulse-chase radiolabeling showed that secreted chimeras lacked the propeptide of cathepsin B. Thus, the results suggest that cathepsin B is first transported to endosomes/lysosomes from where its matured form is retrieved before being secreted, supporting the view that endosome/lysosome-derived cathepsin B contributes to the potential of extracellular proteolysis in the thyroid.

A possible role for the Alzheimer amyloid precursor protein in the regulation of epidermal basal cell proliferation.

The regulation of epidermal growth involves a number of ions, growth factors and cytokines and possibly additional but as yet unknown factors. Here we report on the potential role of the secretory N-terminal domain (sAPP) of the Alzheimer amyloid precursor protein (APP) in the regulation of keratinocyte proliferation. In human skin APP was detectable predominantly in the basal cell layer of the epidermis whereas the immunocytochemical signal in the underlying mesenchymal tissue was very low. Cultured normal human keratinocytes expressed the three APP isoforms 695, 751 and 770 with highest values for the isoforms 751 and 770. HaCaT cells, a spontaneously immortalized human keratinocyte cell line, exhibited almost identical patterns in the expression of the APP isoforms and in the release of endogenous sAPP. In HaCaT cells, recombinant sAPP (sAPPrec) was found to compete with endogenous sAPP for the same binding sites. Binding of sAPPrec was specific and occurred in microdomains of approximately 0.1 to approximately 0.3 microm in diameter. At 10 nM, sAPPrec binding induced a 2- to 4-fold increase in the rate of cell growth. sAPP concentrations in the conditioned media were found to reach 5-20 nM which is in the mitogenic range of sAPPrec. The proliferative effect of sAPP was inhibited by approximately 50% when antisense oligonucleotides directed against the APP mRNA were applied. The predominant expression of

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.

Differentiation of antineutrophil nuclear antibodies in inflammatory bowel and autoimmune liver diseases from antineutrophil cytoplasmic antibodies (p-ANCA) using immunofluorescence microscopy.

Perinuclear antineutrophil cytoplasmic antibodies (p‐ANCA) directed against cytoplasmic proteins of neutrophils have been studied extensively in patients with systemic vasculitides. Recent data indicate that antineutrophil antibodies in sera from patients with chronic inflammatory bowel diseases (IBD) or autoimmune liver disorders, currently called ‘atypical p‐ANCA’, recognize a nuclear target antigen, rendering the term ‘ANCA’ inaccurate. Specific microscopic criteria to distinguish atypical p‐ANCA from p‐ANCA are lacking. We used planar and confocal laser scanning indirect immunofluorescence microscopy to examine the labelling characteristics of ethanol‐, methanol‐ and formaldehyde‐fixed neutrophils by antineutrophil antibodies in 153 serum samples from patients with IBD, autoimmune liver disorders, systemic vasculitides or healthy blood donors. On ethanol‐ or methanol‐fixed neutrophils, multiple intranuclear fluorescent foci together with either a rim‐like peripheral nuclear staining (‘type A’) or a combined cytoplasmic and peripheral nuclear staining (‘type B’) was noted exclusively with atypical p‐ANCA in sera from patients with IBD or autoimmune liver disorders. Intranuclear foci, which probably corresponded to invaginations of the nuclear envelope, were not labelled by p‐ANCA from patients with microscopic polyangiitis or cytoplasmic ANCA (c‐ANCA) from patients with Wegeners granulomatosis. On formaldehyde‐fixed neutrophils, atypical p‐ANCA gave a fine rim‐like staining of the nuclear periphery, whereas ANCA diffusely labelled the cytoplasm. To distinguish reliably between the patterns produced by atypical p‐ANCA or p‐ANCA, particularly p‐ANCA, careful indirect immunofluorescence microscopy on ethanol‐ as well as on formaldehyde‐fixed neutrophils is necessary, with particular emphasis on the presence of multiple intranuclear fluorescent foci.

INTRACELLULAR DISTRIBUTION OF A BIOTIN-LABELED GANGLIOSIDE, GM1, BY IMMUNOELECTRON MICROSCOPY AFTER ENDOCYTOSIS IN FIBROBLASTS

A radioactive and biotin-labeled analogue of GM1 (biotin-GM1) was synthesized which enabled us to analyze its intracellular distribution in the compartments of the endocytic route by electron microscopic immunocytochemistry using thin sections of human skin fibroblasts labeled with gold-conjugated antibiotin antibodies. Metabolic studies with the biotin-GM1 showed its partial degradation to the corresponding GM2 and GM3 derivatives. Further degradation was inhibited by the biotin residue. The distribution of biotin-GM1 after uptake by cells was studied by postembedding labeling techniques. On the plasma membrane the biotin-GM1 was detectable in the form of patches (0.1 μm in diameter), in caveola-like structures and, to a much lesser extent, in coated pits or vesicles. During endocytic uptake, the biotin-GM1 became detectable in organelles identified as late endosomes and lysosomes. The intracellular distribution of the biotin-GM1 was compared to the localization of the EGF receptor in EGF-stimulated fibroblasts. Both the biotin-GM1 and the EGF receptor were transported to intraendosomal and intralysosomal membranes, indicating that both membrane constituents follow the same pathway of endocytosis. Our observations show that biotin-GM1 can be successfully incorporated into the plasma membrane and be used as a tool for morphological detection of its pathway to lysosomes. (J Histochem Cytochem 47:1005–1014, 1999)