4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer's disease

The amyloid β-peptide (Aβ) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer’s disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Aβ and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Aβ can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Aβ might have a stronger (or weaker) inhibitory effect on the proteasome than does native Aβ. We therefore also investigated the proteasome inhibitory action of Aβ _1–40 (a peptide comprising the first 40 residues of Aβ) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H₂O₂ modification of Aβ _1–40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Aβ _1–40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer’s disease Received 26 September 2000; accepted 26 September 2000     

Pancreatic islet β-cell deficit and glucose intolerance in rats with uninephrectomy

This study was performed to examine the effect of chronic renal impairment and renin-angiotensin system (RAS) activation induced by unilateral nephrectomy (UNX) on the development of pancreatic islet β-cell deficit and glucose intolerance. Sprague-Dawley rats were randomized into three groups: untreated UNX (n = 10), UNX treated with the angiotensin-converting enzyme inhibitor lisinopril (n = 8) and sham operation (n = 10). Blood glucose, serum insulin, renal function and histological changes of kidney and pancreas were examined 8 months postoperation. Compared with the sham rats, UNX rats developed renal impairment, insulin deficiency and glucose intolerance. Histological staining revealed an islet β-cell deficit associated with increased immunoreactivity for angiotensin and angiotensin type 1 receptor in UNX rats. Treatment with lisinopril significantly improved renal dysfunction, hyperglycemia, insulin secretion and islet RAS expression. These data suggest that chronic renal impairment and RAS activation may contribute to islet β-cell loss and glucose intolerance. RAS blockade may therefore prevent these disorders. Received 29 August 2007; received after revision 25 September 2007; accepted 27 September 2007     

Involvement of small Ras GTPases and their effectors in chronic renal disease

The mechanisms involved in the development of renal fibrosis are poorly understood. Small Ras GTPases control cell proliferation, differentiation, cellular growth and apoptosis, with cell-specific expression in the kidney. Cytokines, high glucose medium or advanced glycation end-products activate Ras in different renal cells. Increased Ras activation has been found in experimental tubulointerstitial fibrosis. Transforming growth factor-β1 (TGF-β1) and Ras signalling pathways are close related: TGF-β1 overcomes Ras mitogenic effects, and Ras counteracts TGF-β signalling. However, Ras activation is also an intracellular signal transduction point for several molecules (e.g. TGF-β1) involved in kidney damage. Ras isoforms play different roles in regulating extracellular matrix synthesis in fibroblasts and mesangial cells. These data give evidence for a role for Ras in renal fibrosis, but no reviews are available on the role of p21 Ras in this process. Thus, our goal is to review the role of Ras activation and signalling in renal fibrosis. Received 7 June 2007; received after revision 17 September 2007; accepted 1 October 2007     

Podocalyxin enhances the adherence of cells to platelets

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid. Received 14 August 2007; received after revision 12 September 2007; accepted 13 September 2007     

Interactions of the cell adhesion molecule nectin with transmembrane and peripheral membrane proteins for pleiotropic functions

Cell adhesion molecules (CAMs) have been implicated in the control of a wide variety of cellular processes, such as cell adhesion, polarization, survival, movement, and proliferation. Nectins have emerged as immunoglobulin-like CAMs that participate in calcium-independent cell-cell adhesion by homophilic and heterophilic trans-interactions with nectins and nectin-like molecules. Nectin-based cell-cell adhesion exerts its function independently or in cooperation with other CAMs including cadherins and is essential for the formation of intercellular junctions, including adherens junctions, tight junctions, and puncta adherentia junctions. Nectins cis-interact with integrin α_vβ₃ and platelet-derived growth factor receptor and facilitate their signals to regulate the formation and integrity of intercellular junctions and cell survival. Nectins intracellularly associate with peripheral membrane proteins, including afadin and Par-3. This review focuses on recent progress in understanding the interactions of nectins with other transmembrane and peripheral membrane proteins to exert pleiotropic functions. Received 27 June 2007; received after revision 14 August 2007; accepted 12 September 2007     

Inhibition of hepatitis C virus internal ribosome entry site-mediated translation by an RNA targeting the conserved IIIf domain

Hepatitis C virus (HCV) translation initiation depends on an internal ribosome entry site (IRES). We previously identified an RNA molecule (HH363–10) able to bind and cleave the HCV IRES region. This paper characterizes its capacity to interfere with IRES function. Inhibition assays showed that it blocks IRES activity both in vitro and in a human hepatoma cell line. Although nucleotides involved in binding and cleavage reside in separate regions of the inhibitor HH363–10, further analysis demonstrated the strongest effect to be an intrinsic feature of the entire molecule; the abolishment of either of the two activities resulted in a reduction in its function. Probing assays demonstrate that HH363–10 specifically interacts with the conserved IIIf domain of the pseudoknot structure in the IRES, leading to the inhibition of the formation of translationally competent 80S particles. The combination of two inhibitory activities targeting different sequences in a chimeric molecule may be a good strategy to avoid the emergence of resistant viral variants. Received 26 July 2007; received after revision 24 September 2007; accepted 26 September 2007     

Surface molecules regulating rolling and adhesion to endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interaction

The extravasation of leukocytes and tumor cells is a multi-step process with the involvement of various adhesion molecules depending on the three steps rolling, adhesion, and diapedesis. We have developed an in vitro model, by which we investigated the rolling and adhesion of neutrophil granulocytes and MDA-MB-468 human breast carcinoma cells to lung endothelial cells under physiological flow-conditions. We found that norepinephrine had an inhibitory function on the fMLP-promoted adhesion of neutrophil granulocytes due to a down-regulation of β2-integrin. Furthermore, neutrophil granulocytes serve as linking cells for the interaction of the MDA-MB-468 cells with the endothelium, which are both β2-integrin negative, but express the β2-integrin ligand ICAM-1. In addition, we show here that N-cadherin is up-regulated on the endothelial cells and on neutrophil granulocytes in response to fMLP. This up-regulation resulted in a significant increase of adherent MDA-MB-468 cells, which are also N-cadherin positive. Received 3 September 2007; received after revision 17 October 2007; accepted 22 October 2007     

"Add-on" domains of<Emphasis Type="Italic"> Drosophila</Emphasis> β1,4-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminyltransferase B in the stem region and its pilot protein

The glycolipid specific Drosophila melanogaster β1,4-N-acetylgalactosaminyltransferase B (β4GalNAcTB) depends on a zinc finger DHHC protein family member named GalNAcTB pilot (GABPI) for activity and translocation to the Golgi. The six-membrane spanning protein actually lacks the cysteine in the cytoplasmic DHHC motif, displaying DHHS instead. Here we show that the whole conserved region around the DHHS sequence, which is essential for palmitoylation in DHHC proteins, is not required for GABPI to interact with β4GalNAcTB. In contrast, the two luminal loops between transmembrane domain 3–4 and 5–6 contain conserved amino acids, which are crucial for activity. Besides the dependence on GABPI, β4GalNAcTB requires its exceptional short stem region for activity. A few hydrophobic amino acids positioned close to the transmembrane domain are essential for the interaction with GABPI. Along with its catalytic domain, β4GalNAcTB, thus, requires an area in its own stem region and two small luminal loops of GABPI as "add-on" domains. Moreover, some inactive GABPI mutants could be rescued by fusion with β4GalNAcTB, indicating their importance in direct GABPI-β4GalNAcTB interaction.     

A molecular pin to study the dynamics of β-barrel formation in pore-forming toxins on erythrocytes: a sliding model

γ-Hemolysins are pore-forming toxins which develop from water-soluble monomers by combining two different ‘albeit homologous’ proteins. They form oligomeric pores in both cell and model membranes by undergoing a still poorly understood conformational rearrangement in the stem region. The stem is formed by three β-strands, folded onto the core of the soluble protein and completely extended in the pore. We propose a new model to explain such a process. Seven double-cysteine mutants were developed by inserting one cysteine on the stretch that links the β-hairpin to the core of the protein and another on different positions along the β-strands. The membrane bound protein was blocked in a non-lytic state by S–S bond formation. Six mutants were oxidized as inactive intermediates, but became active after adding DTT. These results demonstrate that the stem extension can be temporarily frozen and that the β-barrel formation occurs by β-strand concerted step-by-step sliding. Received 22 October 2007; received after revision 15 November 2007; accepted 19 November 2007     

Roles for CCN2 in normal physiological processes

CCN2, also known as connective tissue growth factor, is a member of the CCN (CCN1–6) family of modular matricellular proteins. Analysis of CCN2 function in vivo has focused primarily on its key role as a mediator of excess ECM synthesis in multiple fibrotic diseases. However, CCN2 and related family members are widely expressed during development. Recent studies using new genetic models are revealing that CCN2 has essential roles in the development of many tissues. This review focuses on current and emerging data on CCN2 and its functions in chondrogenesis and angiogenesis, and on new studies showing that CCN2 has essential functions during embryonic and postnatal development in a number of epithelial tissues.     

Emerging roles of the oxysterol-binding protein family in metabolism, transport, and signaling

OSBP (oxysterol-binding protein) and ORPs (OSBP-related proteins) constitute an enigmatic eukaryotic protein family that is united by a signature domain that binds oxysterols, sterols, and possibly other hydrophobic ligands. The human genome contains 12 OSBP/ORP family members genes, while that of the budding yeast Saccharomyces cerevisiae encodes seven OSBP homologues (Osh). Of these, Osh4 (also referred to as Kes1) has been the most widely studied to date. Recently, three-dimensional crystal structures of Osh4 with and without sterols bound within the core of the protein were determined. The core consists of 19 anti-parallel β-sheets that form a near-complete β-barrel. Recent work has suggested that Osh proteins facilitate the non-vesicular transport of sterols in vivo and in vitro, while other evidence supports a role for Osh proteins in the regulation of vesicular transport and lipid metabolism.This article will review recent advances in the study of ORP/Osh proteins and will discuss future research issues regarding the ORP/Osh family. Received 17 July 2007; received after revision 14 August 2007; accepted 12 September 2007     

Sphingolipid metabolism and its role in the skeletal tissues

The regulators affecting skeletal tissue formation and its maintenance include a wide array of molecules with very diverse functions. More recently, sphingolipids have been added to this growing list of regulatory molecules in the skeletal tissues. Sphingolipids are integral parts of various lipid membranes present in the cells and organelles. For a long time, these macromolecules were considered as inert structural elements. This view, however, has radically changed in recent years as sphingolipids are now recognized as important second messengers for signal-transduction pathways that affect cell growth, differentiation, stress responses and programmed death. In the current review, we discuss the available data showing the roles of various sphingolipids in three different skeletal cell types—chondrocytes in cartilage and osteoblasts and osteoclasts in bone. We provide an overview of the biology of sphingomyelin phosphodiesterase 3 (SMPD3), an important regulator of sphingolipid metabolism in the skeleton. SMPD3 is localized in the plasma membrane and has been shown to cleave sphingomyelin to generate ceramide, a bioactive lipid second messenger, and phosphocholine, an essential nutrient. SMPD3 deficiency in mice impairs the mineralization in both cartilage and bone extracellular matrices leading to severe skeletal deformities. A detailed understanding of SMPD3 function may provide a novel insight on the role of sphingolipids in the skeletal tissues.     

The distribution of α2β1, α3β1 and α6β4 integrins identifies distinct subpopulations of basal keratinocytes in the outer root sheath of the human anagen hair follicle

The human hair follicle is composed of different concentric compartments, which reflect different programmes of differentiation. Using monoclonal antibodies against α2β1 and α3β1 integrins we demonstrated a shift in their expression, from a basolateral distribution in the basal cells of the lower outer root sheath, to an apicolateral expression in the upper outer root sheath, as in epidermis. This shift takes place in a transition zone, localized to the midpart of the follicle. The distinct basolateral distribution of α2β1 and α3β1 integrins in the lower portion of the outer root sheath coincides with the presence of basal cell protrusions and is probably linked to the presence of the vitreous membrane which surrounds the bottom part of the anagen human hair follicle. Moreover, we showed that the expression of α6β4 integrin is discontinuous along the hair follicle and coincides with that of laminin 5. Together these results establish that within a given compartment – namely the outer root sheath – several domains can be clearly identified, which probably reflect the onset of successive differentiation pathways along the hair follicle. Received 17 January 1997; received after revision 18 February 1997; accepted 24 February 1997     

The prolyl oligopeptidase family

A group of serine peptidases, the prolyl oligopeptidase family, cannot hydrolyze peptides containing more than about 30 residues. This group is unrelated to the classical trypsin and subtilisin families, and includes dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B, in addition to the prototype prolyl oligopeptidase. The recent crystal structure determination of prolyl oligopeptidase (80 kDa) has shown that the enzyme contains a peptidase domain with an α/β hydrolase fold, and its catalytic triad is covered by the central tunnel of an unusual seven-bladed β-propeller. This domain operates as a gating filter, excluding large, structured peptides from the active site. The binding mode of substrates and the catalytic mechanism differ from that of the classical serine peptidases in several features. The members of the family are important targets of drug design. Prolyl oligopeptidase is involved in amnesia, depression and blood pressure control, dipeptidyl peptidase IV in type 2 diabetes and oligopeptidase B in trypanosomiasis. Received 8 August 2001; received after revision 19 September 2001; accepted 21 September 2001     

Molecular mechanisms of nitrosative stress-mediated protein misfolding in neurodegenerative diseases

Nitrosative and oxidative stress, associated with the generation of excessive reactive oxygen or nitrogen species, are thought to contribute to neurodegenerative disorders. Many such diseases are characterized by conformational changes in proteins that result in their misfolding and aggregation. Accumulating evidence implies that at least two pathways affect protein folding: the ubiquitin-proteasome system (UPS) and molecular chaperones. Normal protein degradation by the UPS can prevent accumulation of aberrantly folded proteins. Molecular chaperones – such as protein-disulfide isomerase, glucose-regulated protein 78, and heat shock proteins – can provide neuroprotection from aberrant proteins by facilitating proper folding and thus preventing their aggregation. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. Here, we present evidence for the hypothesis that nitric oxide contributes to degenerative conditions by S-nitrosylating specific chaperones or UPS proteins that would otherwise prevent accumulation of misfolded proteins. Received 5 December 2006; received after revision 7 February 2007; accepted 15 March 2007     

Glycoconjugates of the intestinal goblet cells of four cyprinids

The aim of this work was to show differences in the terminal and subterminal sugar composition of carbohydrate chains of glycoconjugates produced by the goblet cells of the intestines of four cyprinids. We analysed intestines of two herbivorous species – sneep and grass carp – and two omnivorous ones – chub and common carp. We compared four intestinal regions of every studied species. In every region, the presence of neutral and acidic glycoconjugates was confirmed. The smallest amount of acidic glycoconjugates was present in the second region of sneep intestine. Sulphated glycoconjugates were absent in the third and fourth region of chub intestine. Lectin histochemistry provided evidence for the presence of β-D-galactose, α-N-acetylgalactosamine, β-N-acetylglucosamine and sialic acids. Additionally, the occurrence of α-L-fucose in the goblet cells of chub, grass carp and sneep was confirmed. We tried to correlate the patern of glycoconjugate glycosylation with feeding habits of the studied fishes. Received 1 July 2002; received after revision 8 August 2002; accepted 19 August 2002 RID="*" ID="*"Corresponding author.     

Nodal signals pattern vertebrate embryos

Vertebrate embryonic patterning requires several conserved inductive signals–including Nodal, Bmp, Wnt and Fgf signals. Nodal, which is a member of the transforming growth factor β (TGFβ) superfamily, activates a signal transduction pathway that is similar to that of other TGFβ members. Nodal genes, which have been identified in numerous vertebrate species, are expressed in specific cell types and tissues during embryonic development. Nodal signal transduction has been shown to play a pivotal role in inducing and patterning mesoderm and endoderm, and in regulating neurogenesis and left-right axis asymmetry. Antagonists, which act at different steps in the Nodal signal transduction pathway, have been shown to tightly modulate the inductive activity of Nodal. Received 20 October 2005; received after revision 15 November 2005; accepted 25 November 2005     

Progesterone regulation of implantation-related genes: new insights into the role of oestrogen

Genomic profiling was performed on explants of late proliferative phase human endometrium after 24-h treatment with progesterone (P) or oestradiol and progesterone (17β-E₂+P) and on explants of menstrual phase endometrium treated with 17β-E₂+P. Gene expression was validated with real-time PCR in the samples used for the arrays, in endometrium collected from early and mid-secretory phase endometrium, and in additional experiments performed on new samples collected in the menstrual and late proliferative phase. The results show that late proliferative phase human endometrium is more responsive to progestins than menstrual phase endometrium, that the expression of several genes associated with embryo implantation (i.e. thrombomodulin, monoamine oxidase A, SPARC-like 1) can be induced by P in vitro, and that genes that are fully dependent on the continuous presence of 17β-E₂ during P exposure can be distinguished from those that are P-dependent to a lesser extent. Therefore, 17β-E₂ selectively primes implantation-related genes for the effects of P. H. Dassen, C. Punyadeera: These authors contributed equally. Received 18 December 2006; received after revision 6 February 2007; accepted 8 March 2007     

Chemical chaperone therapy for GM1-gangliosidosis

We have proposed a chemical chaperone therapy for lysosomal diseases, based on a paradoxical phenomenon that an exogenous competitive inhibitor of low molecular weight stabilizes the target mutant molecule and restores its catalytic activity as a molecular chaperone intracellularly. After Fabry disease experiments, we investigated a new synthetic chaperone compound N-octyl-4-epi-β-valienamine (NOEV) in a G_M1-gangliosidosis model mice. Orally administered NOEV entered the brain through the blood-brain barrier, enhanced β-galactosidase activity, reduced the substrate storage, and clinically improved neurological deterioration. We hope that chemical chaperone therapy will prove useful for some patients with G_M1-gangliosidosis and potentially other lysosomal storage diseases with central nervous system involvement. Received 10 October 2007; received after revision 31 October 2007; accepted 6 November 2007