Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion.

Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel- and cone-like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain. RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

Asi1 is an inner nuclear membrane protein that restricts promoter access of two latent transcription factors.

Stp1 and Stp2 are homologous transcription factors in yeast that are synthesized as latent cytoplasmic precursors with NH2-terminal regulatory domains. In response to extracellular amino acids, the plasma membrane-localized Ssy1-Ptr3-Ssy5 (SPS) sensor endoproteolytically processes Stp1 and Stp2, an event that releases the regulatory domains. The processed forms of Stp1 and Stp2 efficiently target to the nucleus and bind promoters of amino acid permease genes. In this study, we report that Asi1 is an integral component of the inner nuclear membrane that maintains the latent characteristics of unprocessed Stp1 and Stp2. In cells lacking Asi1, full-length forms of Stp1 and Stp2 constitutively induce SPS sensor-regulated genes. The regulatory domains of Stp1 and Stp2 contain a conserved motif that confers Asi1-mediated control when fused to an unrelated DNA-binding protein. Our results indicate that latent precursor forms of Stp1 and Stp2 inefficiently enter the nucleus; however, once there, Asi1 restricts them from binding SPS sensor-regulated promoters. These findings reveal an unanticipated role of inner nuclear membrane proteins in controlling gene expression.

Nucleoporins as components of the nuclear pore complex core structure and Tpr as the architectural element of the nuclear basket.

The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.

Structure and influence on stability and activity of the N-terminal propeptide part of lung surfactant protein C.

Mature lung surfactant protein C (SP-C) corresponds to residues 24-58 of the 21 kDa proSP-C. A late processing intermediate, SP-Ci, corresponding to residues 12-58 of proSP-C, lacks the surface activity of SP-C, and the SP-Ci alpha-helical structure does not unfold in contrast to the metastable nature of the SP-C helix. The NMR structure of an analogue of SP-Ci, SP-Ci(1-31), with two palmitoylCys replaced by Phe and four Val replaced by Leu, in dodecylphosphocholine micelles and in ethanol shows that its alpha-helix vs. that of SP-C is extended N-terminally. The Arg-Phe part in SP-Ci that is cleaved to generate SP-C is localized in a turn structure, which is followed by a short segment in extended conformation. Circular dichroism spectroscopy of SP-Ci(1-31) in microsomal or surfactant lipids shows a mixture of helical and extended conformation at pH 6, and a shift to more unordered structure at pH 5. Replacement of the N-terminal hexapeptide segment SPPDYS (known to constitute a signal in intracellular targeting) of SP-Ci with AAAAAA results in a peptide that is mainly unstructured, independent of pH, in microsomal and surfactant lipids. Addition of a synthetic dodecapeptide, corresponding to the propeptide part of SP-Ci, to mature SP-C results in slower aggregation kinetics and altered amyloid fibril formation, and reduces the surface activity of phospholipid-bound SP-C. These data suggest that the propeptide part of SP-Ci prevents unfolding by locking the N-terminal part of the helix, and that acidic pH results in structural disordering of the region that is proteolytically cleaved to generate SP-C.

Comparative evaluation of FGF-2-, VEGF-A-, and VEGF-C-induced angiogenesis, lymphangiogenesis, vascular fenestrations, and permeability.

Several endothelial growth factors induce both blood and lymphatic angiogenesis. However, a systematic comparative study of the impact of these factors on vascular morphology and function has been lacking. In this study, we report a quantitative analysis of the structure and macromolecular permeability of FGF-2-, VEGF-A-, and VEGF-C-induced blood and lymphatic vessels. Our results show that VEGF-A stimulated formation of disorganized, nascent vasculatures as a result of fusion of blood capillaries into premature plexuses with only a few lymphatic vessels. Ultrastructural analysis revealed that VEGF-A-induced blood vessels contained high numbers of endothelial fenestrations that mediated high permeability to ferritin, whereas the FGF-2-induced blood vessels lacked vascular fenestrations and showed only little leakage of ferritin. VEGF-C induced approximately equal amounts of blood and lymphatic capillaries with endothelial fenestrations present only on blood capillaries, mediating a medium level of ferritin leakage into the perivascular space. No endothelial fenestrations were found in FGF-2-, VEGF-A-, or VEGF-C-induced lymphatic vessels. These findings highlight the structural and functional differences between blood and lymphatic vessels induced by FGF-2, VEGF-A, and VEGF-C. Such information is important to consider in development of novel therapeutic strategies using these angiogenic factors.

Increased intimal hyperplasia and smooth muscle cell proliferation in transgenic mice with heparan sulfate-deficient perlecan.

Smooth muscle cell (SMC) proliferation is a critical process in vascular disease. Heparan sulfate (HS) proteoglycans inhibit SMC growth, but the role of endogenous counterparts in the vessel wall in control of SMC function is not known in detail. Perlecan is the major HS proteoglycans in SMC basement membranes and in vessel wall extracellular matrix (ECM). In this study, transgenic mice with HS-deficient perlecan were analyzed with respect to vascular phenotype and intimal lesion formation. Furthermore, SMC cultures were established and characterized with respect to morphology, immunocytochemical features, proteoglycan synthesis, proliferative capacity, and ECM binding of basic fibroblast growth factor (FGF-2). In vitro, mutant SMCs formed basement membranes with perlecan core protein, but with decreased levels of HS, they showed diminished secretion of HS-containing perlecan into the medium and a defective ECM-binding capacity of FGF-2. In vitro, mutant SMCs showed increased proliferation compared with wild-type cells, and in vivo, enhanced SMC proliferation and intimal hyperplasia were observed after flow cessation of the carotid artery in mutant mice. The results indicate that the endogenous HS side-chains of perlecan contribute to SMC growth control both in vitro and during intimal hyperplasia, possibly by sequestering heparin-binding mitogens such as FGF-2.

Allogenic immune response promotes the accumulation of host-derived smooth muscle cells in transplant arteriosclerosis.

OBJECTIVE: Smooth muscle cells (SMCs) involved in intimal hyperplasia during transplant vasculopathy are derived both from the graft and the host. Here, the role of an allogenic immune response in the accumulation of host-derived SMCs in the neointima was explored. METHODS: Infrarenal aorta was transplanted from female F344 to male Lewis rats with or without immunosuppression by cyclosporine A (CsA). Accumulation of host-derived SMCs and inflammatory cells in the grafts, SMC proliferation, and apoptosis were analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR) for the SRY gene. Finally, SMCs were seeded in an allogenic or isogenic manner after balloon injury to carotid arteries and SMC survival was estimated. RESULTS: Proliferating graft SMCs and infiltrating leukocytes were observed in the intima early after transplantation. In parallel, inflammatory cells and immunoglobulins infiltrated the media and apoptosis of medial SMCs occurred, leading to destruction of this layer. CsA decreased the number of SRY+ SMCs in the lesions, restricted medial destruction, and improved survival of allogenic SMCs after seeding in injured arteries. CONCLUSIONS: Development of intimal thickenings during transplant vasculopathy involves an allogenic immune response, which promotes accumulation of host-derived SMCs and apoptosis of resident graft SMCs.

Small GTP-binding protein Rac is an essential mediator of vascular endothelial growth factor-induced endothelial fenestrations and vascular permeability.

BACKGROUND: Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces both angiogenesis and vascular permeability. Although its angiogenic activity has been well characterized, the signaling pathways of VEGF-induced permeability remain poorly understood. METHODS AND RESULTS: Using the mouse corneal micropocket assay, Miles assay, and a combination of cytochemical, electron microscopic, and biochemical assays, we demonstrate that VEGF-induced vascular leakage partly can be separated from its angiogenic activity. VEGF but not FGF-2 induced capillaries with a highly fenestrated endothelium, a feature linked with increased vascular permeability. A cell-permeable Rac antagonist (TAT-RacN17) converted VEGF-induced, leaky vascular plexuses into well-defined vascular networks. In addition, this Rac mutant blocked formation of VEGF-induced endothelial fenestrations and vascular permeability but only partially inhibited angiogenesis. Studies on endothelial cell cultures further revealed that VEGF stimulated phosphorylation of VEGF receptor-2 (VEGFR-2), leading to activation of Rac as well as increased phosphorylation of phospholipase Cgamma (PLCgamma), protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), and extracellular regulated kinase (Erk1/2). We further found that phosphatidylinositol-3-OH kinase (PI3K) acted upstream of Rac and Akt-eNOS in VEGF/VEGFR-2 signaling. CONCLUSIONS: Our findings indicate that the small GTP-binding protein Rac is a key component in mediation of VEGF-induced vascular permeability but less so in neovascularization. This may have conceptual implications for applying Rac antagonists in treatment and prevention of VEGF-induced vascular leakage and edema in connection with ischemic disorders.

Collagenous Alzheimer amyloid plaque component assembles amyloid fibrils into protease resistant aggregates.

Recently, a novel plaque-associated protein, collagenous Alzheimer amyloid plaque component (CLAC), was identified in brains from patients with Alzheimer's disease. CLAC is derived from a type II transmembrane collagen precursor protein, termed CLAC-P (collagen XXV). The biological function and the contribution of CLAC to the pathogenesis of Alzheimer's disease and plaque formation are unknown. In vitro studies indicate that CLAC binds to fibrillar, but not to monomeric, amyloid beta-peptide (Abeta). Here, we examined the effects of CLAC on Abeta fibrils using assays based on turbidity, thioflavin T binding, sedimentation analysis, and electron microscopy. The incubation of CLAC with preformed Abeta fibrils led to increased turbidity, indicating that larger aggregates were formed. In support of this contention, more Abeta was sedimented in the presence of CLAC, as determined by gel electrophoresis. Moreover, electron microscopy revealed an increased amount of Abeta fibril bundles in samples incubated with CLAC. Importantly, the frequently used thioflavin T-binding assay failed to reveal these effects of CLAC. Digestion with proteinase K or trypsin showed that Abeta fibrils, incubated together with CLAC, were more resistant to proteolytic degradation. Therefore, CLAC assembles Abeta fibrils into fibril bundles that have an increased resistance to proteases. We suggest that CLAC may act in a similar way in vivo.

The N-terminal propeptide of lung surfactant protein C is necessary for biosynthesis and prevents unfolding of a metastable alpha-helix.

The lung surfactant-associated protein C (SP-C) consists mainly of a polyvaline alpha-helix, which is stable in a lipid membrane. However, in agreement with the predicted beta-strand conformation of a polyvaline segment, helical SP-C unfolds and transforms into beta-sheet aggregates and amyloid fibrils within a few days in aqueous organic solvents. SP-C fibril formation and aggregation have been associated with lung disease. Here, we show that in a recently isolated biosynthetic precursor of SP-C (SP-Ci), a 12 residue N-terminal propeptide locks the metastable polyvaline part in a helical conformation. The SP-Ci helix does not aggregate or unfold during several weeks of incubation, as judged by hydrogen/deuterium exchange and mass spectrometry. Hydrogen/deuterium exchange experiments further indicate that the propeptide reduces exchange in parts corresponding to mature SP-C. Finally, in an acidic environment, SP-Ci unfolds and aggregates into amyloid fibrils like SP-C. These data suggest a direct role of the N-terminal propeptide in SP-C biosynthesis.

Expanded substrate screenings of human and Drosophila type 10 17beta-hydroxysteroid dehydrogenases (HSDs) reveal multiple specificities in bile acid and steroid hormone metabolism: characterization of multifunctional 3alpha/7alpha/7beta/17beta/20beta/21-HSD.

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the conversion of 17beta-OH (-hydroxy)/17-oxo groups of steroids, and are essential in mammalian hormone physiology. At present, eleven 17beta-HSD isoforms have been defined in mammals, with different tissue-expression and substrate-conversion patterns. We analysed 17beta-HSD type 10 (17beta-HSD10) from humans and Drosophila, the latter known to be essential in development. In addition to the known hydroxyacyl-CoA dehydrogenase, and 3alpha-OH and 17beta-OH activities with sex steroids, we here demonstrate novel activities of 17beta-HSD10. Both species variants oxidize the 20beta-OH and 21-OH groups in C21 steroids, and act as 7beta-OH dehydrogenases of ursodeoxycholic or isoursodeoxycholic acid (also known as 7beta-hydroxylithocholic acid or 7beta-hydroxyisolithocholic acid respectively). Additionally, the human orthologue oxidizes the 7alpha-OH of chenodeoxycholic acid (5beta-cholanic acid, 3alpha,7alpha-diol) and cholic acid (5beta-cholanic acid). These novel substrate specificities are explained by homology models based on the orthologous rat crystal structure, showing a wide hydrophobic cleft, capable of accommodating steroids in different orientations. These properties suggest that the human enzyme is involved in glucocorticoid and gestagen catabolism, and participates in bile acid isomerization. Confocal microscopy and electron microscopy studies reveal that the human form is localized to mitochondria, whereas Drosophila 17beta-HSD10 shows a cytosolic localization pattern, possibly due to an N-terminal sequence difference that in human 17beta-HSD10 constitutes a mitochondrial targeting signal, extending into the Rossmann-fold motif.

Stabilization of discordant helices in amyloid fibril-forming proteins.

Several proteins and peptides that can convert from alpha-helical to beta-sheet conformation and form amyloid fibrils, including the amyloid beta-peptide (Abeta) and the prion protein, contain a discordant alpha-helix that is composed of residues that strongly favor beta-strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but Abeta apparently lacks such interactions. The helical propensity of the Abeta discordant region (K16LVFFAED23) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the alpha-helical content of Abeta(12-28) and reduces aggregation and fibril formation of Abeta(1-40), Abeta(12-28), Abeta(12-24), and Abeta(14-23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on Abeta secondary structure or fibril formation. These data suggest that in free Abeta, the discordant alpha-helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.

Shortened life span, bradycardia, and hypotension in mice with targeted expression of an Igf2 transgene in smooth muscle cells.

IGF2 is known to affect the normal development and pathology of the cardiovascular system. We previously created mutant mice with targeted expression of an Igf2 transgene in the smooth muscle cells and showed that these mice spontaneously develop aortic intimal cushions. In the present work, we provide a general description of the phenotype of two independent lines of heterozygous transgenics. These mice showed organomegaly and a shortened life span. The latter trait was stronger in the line with a relatively more marked organomegaly and more pronounced in males than females in both lines. Postmortem histology revealed gross abnormalities of the cardiac architecture, suggesting that transgenic mice may accumulate lethal cardiovascular defects. Accordingly, apparently normal transgenic mice had mild cardiomegaly, an enlarged left ventricle, bradycardia, and hypotension. These observations are discussed in the light of the proposed therapeutic use of IGF2 in human cardiac diseases.

Smooth-muscle progenitor cells of bone marrow origin contribute to the development of neointimal thickenings in rat aortic allografts and injured rat carotid arteries.

This study indicates that circulating progenitors of bone marrow origin give rise to cells with smooth muscle-like properties during formation of neointimal thickenings in the arterial wall after allotransplantation and after balloon injury. A segment of abdominal aorta was transplanted from female F344 to male LEW rats, and the grafts were analyzed for male cells by using the gene as a marker. Immunostaining demonstrated that CD45-positive leukocytes made up 35-45% of the neointimal cells during the 8-week period examined. Concurrently, up to 70% of the neointimal cells were of host origin, as shown by real-time polymerase chain reaction for the gene (Y chromosome). This suggests that the neointima contained host cells also of noninflammatory character. Accordingly, many cells positive for smooth-muscle alpha-actin were detected in this layer. To explore the possible bone marrow origin of allograft cells, female LEW rats were irradiated and substituted with bone marrow from male LEW rats. Subsequently, the animals received an aortic transplant from female F344 rats or were exposed to a balloon injury of the carotid artery. Immunostaining and real-time polymerase chain reaction confirmed the above findings, but the fractions of leukocytes and -positive cells were lower in the carotids than in the allografts. Combined primed in situ labeling and immunostaining verified that not only inflammatory but also smooth muscle-like cells of male origin appeared in the vessel wall in both situations. These observations suggest that the smooth-muscle cells that participate in the development of neointimal lesions during vascular disease may, in part, originate from circulating progenitors.

Caveolae and cholesterol distribution in vascular smooth muscle cells of different phenotypes.

Vascular smooth muscle cells (SMCs) grown in primary culture are converted from a contractile to a synthetic phenotype. This includes a marked morphological reorganization, with loss of myofilaments and formation of a large ER-Golgi complex. In addition, the number of cell surface caveolae is distinctly reduced and the handling of lipoprotein-derived cholesterol changed. Here we used filipin as a marker to study the distribution of cholesterol in SMCs by electron microscopy. In contractile cells, filipin-sterol complexes were preferentially found in caveolae and adjacent ER cisternae (present in both leaflets of the membranes). After exposure to LDL or cholesterol, labeling with filipin was increased both in membrane organelles and in the cytoplasm. In contrast, treatment with mevinolin (a cholesterol synthesis inhibitor) or beta-cyclodextrin (a molecule that extracts cholesterol from cells) decreased the reaction with filipin but did not affect the close relation between the ER and the cell surface. In synthetic cells, filipin-sterol complexes were diffusely spread in the plasma membrane and the strongest cytoplasmic reaction was noted in endosomes/lysosomes, both under normal conditions and after incubation with LDL or cholesterol. On the basis of the present findings, we propose a mechanism for direct exchange of cholesterol between the plasma membrane and the ER and more active in contractile than in synthetic SMCs.

The Arctic amyloid-ß precursor protein (AßPP) mutation results in distinct plaques and accumulation of N- and C-truncated Aß.

The Arctic (p. E693G) mutation in the amyloid-ß precursor protein (AßPP) facilitates amyloid-ß (Aß) protofibril formation and generates clinical symptoms of Alzheimer's disease (AD). Here, molecular details of Aß in post mortem brain were investigated with biochemical and morphological techniques. The basic structure of Arctic plaques resembled cotton wool plaques. However, they appeared ring-formed with Aß42-specific antibodies, but were actually targetoid, since the periphery and center of many parenchymal Aß deposits stained differently with mid-domain, N- and C-terminal Aß antibodies. Aß fibrils were similar in shape, albeit shorter than in sporadic AD brain, when examined by electron microscopy. Aßwild-type and Aßarctic codeposited and parenchymal deposits were highly enriched in both N- and C-terminally truncated Aß. In contrast, cerebral amyloid angiopathy (CAA) contained a substantial amount of Aß1-40. The absence of plaques with cores of fibrillary Aß might be due to the scarcity of full-length Aß, although other mechanisms could be involved. Our findings are discussed in relation to mechanisms and relevance of amyloid formation and to the clinical features of AD.

Inner nuclear membrane proteins Asi1, Asi2, and Asi3 function in concert to maintain the latent properties of transcription factors Stp1 and Stp2.

In yeast the homologous transcription factors Stp1 and Stp2 are synthesized as latent cytoplasmic precursors with N-terminal regulatory domains. In response to extracellular amino acids the regulatory domains are endoproteolytically excised by the plasma membrane-localized SPS sensor. The processed forms of Stp1 and Stp2 efficiently enter the nucleus and induce expression of amino acid permease genes. We recently reported that the inner nuclear membrane protein Asi1 is required to prevent unprocessed forms of Stp1 and Stp2, which ectopically enter the nucleus, from binding SPS sensor-regulated promoters. Here we show that Asi3, an Asi1 homolog, and Asi2 are integral proteins of the inner nuclear membrane that function in concert with Asi1. In cells lacking any of the three Asi proteins, unprocessed full-length forms of Stp1 and Stp2 constitutively induce SPS sensor-regulated genes. Our results demonstrate that the Asi proteins ensure the fidelity of SPS sensor signaling by maintaining the dormant, or repressed state, of gene expression in the absence of inducing signals. This study documents additional components of a novel mechanism controlling transcription in eukaryotic cells.