Structure-function analysis of human stomatin: A mutation study.

Stomatin is an ancient, widely expressed, oligomeric, monotopic membrane protein that is associated with cholesterol-rich membranes/lipid rafts. It is part of the SPFH superfamily including stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins and erlins. Biochemical features such as palmitoylation, oligomerization, and hydrophobic "hairpin" structure show similarity to caveolins and other integral scaffolding proteins. Recent structure analyses of the conserved PHB/SPFH domain revealed amino acid residues and subdomains that appear essential for the structure and function of stomatin. To test the significance of these residues and domains, we exchanged or deleted them, expressed respective GFP-tagged mutants, and studied their subcellular localization, molecular dynamics and biochemical properties. We show that stomatin is a cholesterol binding protein and that at least two domains are important for the association with cholesterol-rich membranes. The conserved, prominent coiled-coil domain is necessary for oligomerization, while association with cholesterol-rich membranes is also involved in oligomer formation. FRAP analyses indicate that the C-terminus is the dominant entity for lateral mobility and binding site for the cortical actin cytoskeleton.

Stomatin interacts with GLUT1/SLC2A1, band 3/SLC4A1, and aquaporin-1 in human erythrocyte membrane domains.

The widely expressed, homo-oligomeric, lipid raft-associated, monotopic integral membrane protein stomatin and its homologues are known to interact with and modulate various ion channels and transporters. Stomatin is a major protein of the human erythrocyte membrane, where it associates with and modifies the glucose transporter GLUT1; however, previous attempts to purify hetero-oligomeric stomatin complexes for biochemical analysis have failed. Because lateral interactions of membrane proteins may be short-lived and unstable, we have used in situ chemical cross-linking of erythrocyte membranes to fix the stomatin complexes for subsequent purification by immunoaffinity chromatography. To further enrich stomatin, we prepared detergent-resistant membranes either before or after cross-linking. Mass spectrometry of the isolated, high molecular, cross-linked stomatin complexes revealed the major interaction partners as glucose transporter-1 (GLUT1), anion exchanger (band 3), and water channel (aquaporin-1). Moreover, ferroportin-1 (SLC40A1), urea transporter-1 (SLC14A1), nucleoside transporter (SLC29A1), the calcium-pump (Ca-ATPase-4), CD47, and flotillins were identified as stomatin-interacting proteins. These findings are in line with the hypothesis that stomatin plays a role as membrane-bound scaffolding protein modulating transport proteins.

Interferon gamma-induced human guanylate binding protein 1 inhibits mammary tumor growth in mice.

Interferon gamma (IFN-gamma) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-gamma are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-gamma-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-gamma-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-gamma-induced antitumoral defense system.

Palisade endings are present in canine extraocular muscles and have a cholinergic phenotype.

Classical proprioceptors, like Golgi tendon organs and muscle spindles are absent in the extraocular muscles (EOMs) of most mammals. Instead, a nerve end organ was detected in the EOMs of each species including sheep, cat, rabbit, rat, monkey, and human examined so far: the palisade ending. Until now no clear evidence appeared that palisade endings are also present in canine EOMs. Here, we analyzed dog EOMs by confocal laser scanning microscopy, 3D reconstruction, and transmission electron microscopy. In EOM wholemount preparations stained with antibodies against neurofilament and synaptophysin we could demonstrate typical palisade endings. Nerve fibers coming from the muscle extend into the tendon. There, the nerve fibers turn 180 degrees and return to branch into preterminal axons which establish nerve terminals around a single muscle fiber tip. Fine structural analysis revealed that each palisade ending in dog EOMs establish nerve terminals on the tendon. In some palisade endings we found nerve terminals contacting the muscle fiber as well. Such neuromuscular contacts have a basal lamina in the synaptic cleft. By using an antibody against choline acetyltransferase (ChAT) we proved that canine palisade endings are ChAT-immunoreactive. This study shows that palisade endings are present in canine EOMs. In line with prior findings in cat and monkey, palisade endings in dog have a cholinergic phenotype.

Ultrastructural and molecular biologic comparison of classic proprioceptors and palisade endings in sheep extraocular muscles.

PURPOSE: To analyze and compare the structural and molecular features of classic proprioceptors like muscle spindles and Golgi tendon organs (GTOs) and putative proprioceptors (palisade endings) in sheep extraocular muscle (EOMs). METHODS: The EOMs of four sheep were analyzed. Frozen sections or wholemount preparations of the samples were immunohistochemically labeled and analyzed by confocal laser scanning microscopy. Triple labeling with different combinations of antibodies against neurofilament, synaptophysin, and choline acetyltransferase (ChAT), as well as alpha-bungarotoxin and phalloidin, was performed. Microscopic anatomy of the nerve end organs was analyzed by transmission electron microscopy. RESULTS: The microscopic anatomy demonstrated that muscle spindles and GTOs had a perineural capsule and palisade endings a connective tissue capsule. Sensory nerve terminals in muscle spindles and GTOs contained only a few vesicles, whereas palisade nerve terminals were full of clear vesicles. Likewise, motor terminals in the muscle spindles' polar regions were full of clear vesicles. Immunohistochemistry showed that sensory nerve fibers as well as their sensory nerve terminals in muscle spindles and GTOs were ChAT-negative. Palisade endings were supplied by ChAT-positive nerve fibers, and the palisade complexes including palisade nerve terminals were also ChAT-immunoreactive. Motor terminals in muscle spindles were ChAT and alpha-bungarotoxin positive. CONCLUSIONS: The present study demonstrated in sheep EOMs that palisade endings are innervated by cholinergic axons exhibiting characteristics typical of motoneurons, whereas muscle spindles (except the polar regions) and GTOs are supplied by noncholinergic axons. These results raise the question of whether palisade endings are candidates for proprioceptors in EOMs.

Promoter complex in the central part of the mouse mammary tumor virus long terminal repeat.

Unique among the retroviruses, mouse mammary tumor virus (MMTV) carries, in addition to the usual long terminal repeat (LTR) promoter, another promoter, P2, which is located in the central part of the proviral U3 sequence, within the LTR open reading frame (ORF). Using an in vitro reporter system based on a sensitive luciferase expression assay, we investigated the regulation of the P2 promoter in the context of the Mtv-2 and Mtv-8 genomes. Irrespective of the genomic source, the activity of the P2 promoter is regulated by a downstream-located enhancer and an upstream-located negative regulatory element (NRE), the activity of which overrides the activator. During this study, we unexpectedly detected another independent neighboring promoter that we called P3. The novel P3 promoter does not seem to be controlled by any NRE but is influenced by the same enhancer that modulates the P2 promoter. The respective transcription starts of the two promoters located in this tight cluster are only 61 bases apart. The transcripts originating from this promoter complex carry the same first intron, which is bound by canonical splice donor and splice acceptor sites located in the LTR. One novel doubly spliced transcript carrying a 459-nucleotide-long ORF was detected in several MMTV-carrying murine cells and could be successfully expressed in murine cells as a His-tagged fusion product. The novel viral protein, the function of which remains to be elucidated, has an apparent molecular mass of 20 kDa.

Abundant authentic MMTV-Env production from a recombinant provirus lacking the major LTR promoter.

As for all retroviruses, the env mRNA is thought to be a singly spliced product of the full-length transcript from the P1 promoter in the MMTV provirus. However, we show that envelope proteins can be produced in an inducible manner in the absence of the P1 promoter from an otherwise complete provirus. Furthermore, we demonstrate in both reporter assays and the proviral context that the R region is necessary for protein production in transiently transfected cells and in a number of independent, stably transfected cell clones. Using 5' RACE, we show that a sequence within the R region functions as a TATA less initiator. The most distal part of the 5' LTR (first 804 bases of the U3 region) is required for the activity of the R-initiator element only when the provirus is integrated. Transfection with a full-length proviral DNA carrying a deletion of P1 in the 5' LTR resulted in the establishment of stable cell clones able to produce Env in a dexamethasone-dependent manner but not infectious virions. We therefore conclude that in the absence of P1, R can drive transcription of the spliced env mRNA but not genomic viral RNA.