Low density lipoprotein receptor-related protein 1 couples ß1 integrin activation to degradation.

Low density lipoprotein receptor-related protein (LRP) 1 modulates cell adhesion and motility under normal and pathological conditions. Previous studies documented that LRP1 binds several integrin receptors and mediates their trafficking to the cell surface and endocytosis. However, the mechanism by which LRP1 may regulate integrin activation remains unknown. Here we report that LRP1 promotes the activation and subsequent degradation of ß1 integrin and thus supports cell adhesion, spreading, migration and integrin signaling on fibronectin. LRP1 interacts with surface ß1 integrin, binds the integrin activator kindlin2 and stimulates ß1 integrin-kindlin2 complex formation. Specifically, serine 76 in the LRP1 cytoplasmic tail is crucial for the interaction with kindlin2, ß1 integrin activation and cell adhesion. Interestingly, a loss of LRP1 induces the accumulation of several integrin receptors on the cell surface. Following internalization, intracellular trafficking of integrins is driven by LRP1 in a protein kinase C- and class II myosin-dependent manner. Ultimately, LRP1 dictates the fate of endocytosed ß1 integrin by directing it down the pathway of lysosomal and proteasomal degradation. We propose that LRP1 mediates cell adhesion by orchestrating a multi-protein pathway to activate, traffic and degrade integrins. Thus, LRP1 may serve as a focal point in the integrin quality control system to ensure a firm connection to the extracellular matrix.

Cofilin, a hypoxia-regulated protein in murine lungs identified by 2DE: role of the cytoskeletal protein cofilin in pulmonary hypertension.

Chronic alveolar hypoxia induces vascular remodeling processes in the lung resulting in pulmonary hypertension (PH). However, the mechanisms underlying pulmonary remodeling processes are not fully resolved yet. To investigate functional changes occurring during hypoxia exposure we applied 2DE to compare protein expression in lungs from mice subjected to 3 h of alveolar hypoxia and those kept under normoxic conditions. Already after this short-time period several proteins were significantly regulated. Subsequent analysis by MALDI-MS identified cofilin as one of the most prominently upregulated proteins. The regulation was confirmed by western blotting and its cellular localization was determined by immunohisto- and immunocytochemistry. Interestingly, enhanced cofilin serine 3 phosphorylation was observed after short-term and after chronic hypoxia-induced PH in mice, in pulmonary arterial smooth muscle cells (PASMC) from monocrotaline-induced PH in rats, in lungs of idiopathic pulmonary arterial hypertension patients and in hypoxic or platelet-derived growth factor BB-treated human PASMC. Furthermore, elevated cofilin phosphorylation was attenuated by curative treatment of monocrotaline-induced PH in rats and hypoxia-induced PH in mice with the PDGF-BB receptor antagonist imatinib. In conclusion, short-term hypoxic exposure induced prominent changes in lung protein regulation. These very early changes allowed us to identify potential triggers of PH. Thus, respective 2DE analysis can lead to the identification of new target proteins for the possible treatment of PH.

Comparative proteomic analysis of lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and lung transplant donor lungs.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease for which no effective therapy exists to date. To identify the molecular mechanisms underlying IPF, we performed comparative proteome analysis of lung tissue from patients with sporadic IPF (n = 14) and human donor lungs (controls, n = 10) using two-dimensional gel electrophoresis and MALDI-TOF-MS. Eighty-nine differentially expressed proteins were identified, from which 51 were up-regulated and 38 down-regulated in IPF. Increased expression of markers for the unfolded protein response (UPR), heat-shock proteins, and DNA damage stress markers indicated a chronic cell stress-response in IPF lungs. By means of immunohistochemistry, induction of UPR markers was encountered in type-II alveolar epithelial cells of IPF but not of control lungs. In contrast, up-regulation of heat-shock protein 27 (Hsp27) was exclusively observed in proliferating bronchiolar basal cells and associated with aberrant re-epithelialization at the bronchiolo-alveolar junctions. Among the down-regulated proteins in IPF were antioxidants, members of the annexin family, and structural epithelial proteins. In summary, our results indicate that IPF is characterized by epithelial cell injury, apoptosis, and aberrant epithelial proliferation.

Eimeria bovis-induced modulation of the host cell proteome at the meront I stage.

The proteome of Eimeria bovis meront I-carrying host cells was analyzed by two-dimensional gel electrophoresis (2DE) at 14 days p.i. and compared to non-infected control cells. A total of 221 protein spots were modulated in their abundance in E. bovis-infected host cells and were subsequently analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectometry (MALDI-TOF-MS). These analyses identified 104 proteins in total with 25 host cell proteins being up-regulated and 79 proteins being down-regulated in E. bovis-infected host cells. Moreover, 20 newly expressed proteins were identified exclusively in E. bovis-infected host cells and were most likely of parasite origin. Parasite-induced differences in protein abundance concerned distinct functional categories, with most proteins being involved in host cell metabolism, cell structure, protein fate and gene transcription. Some of the modulated molecules also indicated regulatory processes on the level of host cell stress response (HSP70, HSP90), host cell apoptosis (caspase 8) and actin elongation/depolymerization (α-actinin-1, gelsonin, tropomodulin-3, transgelin). Since merozoites I were already released shortly after cell sampling, the current data reflect the situation at the end of first merogony. This is the first proteomic approach on E. bovis-infected host cells that was undertaken to gain a rather broad insight into Eimeria-induced host cell modulation. The data processed in this investigation should provide a useful basis for more detailed analyses concerning Eimeria-host cell interactions.

Proteomic changes in maize roots after short-term adjustment to saline growth conditions.

It is of fundamental importance to understand adaptation processes leading to salt resistance. The initial effects on maize roots in the first hour after the adjustment to saline conditions were monitored to elucidate initial responses. The subsequent proteome change was monitored using a 2-D proteomic approach. We found several new salt-inducible proteins, whose expression has not been previously reported to be modulated by salt. A set of phosphoproteins in maize was detected but only ten proteins were phosphorylated and six proteins were dephosphorylated after the application of 25 mM NaCl for 1 h. Some of the phosphorylated maize proteins such as fructokinase, UDP-glucosyl transferase BX9, and 2-Cys-peroxyredoxine were enhanced, whereas an isocitrate-dehydrogenase, calmodulin, maturase, and a 40-S-ribosomal protein were dephosphorylated after adjustment to saline conditions. The initial reaction of the proteome and phosphoproteome of maize after adjustment to saline conditions reveals members of sugar signalling and cell signalling pathways such as calmodulin, and gave hint to a transduction chain which is involved in NaCl-induced signalling. An alteration of 14-3-3 proteins as detected may change plasma membrane ATPase activity and cell wall growth regulators such as xyloglucane endotransglycosylase were also found to be changed immediately after the adjustment to salt stress.

Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence.

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase-mediated effect on post-stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488-4496). In the present study, the global function of ClpC in physiology, metabolism, and late-stationary phase survival was examined using DNA microarrays and 2-D PAGE followed by MALDI-TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose-phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell-associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late-stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.

Fhl-1, a new key protein in pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a severe disease with a poor prognosis. Different forms of PH are characterized by pronounced vascular remodeling, resulting in increased vascular resistance and subsequent right heart failure. The molecular pathways triggering the remodeling process are poorly understood. We hypothesized that underlying key factors can be identified at the onset of the disease. Thus, we screened for alterations to protein expression in lung tissue at the onset of PH in a mouse model of hypoxia-induced PH. METHODS AND RESULTS: Using 2-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight analysis, we identified 36 proteins that exhibited significantly altered expression after short-term hypoxic exposure. Among these, Fhl-1, which is known to be involved in muscle development, was one of the most prominently upregulated proteins. Further analysis by immunohistochemistry, Western blot, and laser-assisted microdissection followed by quantitative polymerase chain reaction confirmed the upregulation of Fhl-1, particularly in the pulmonary vasculature. Comparable upregulation was confirmed (1) after full establishment of hypoxia-induced PH, (2) in 2 rat models of PH (monocrotaline-treated and hypoxic rats treated with the vascular endothelial growth factor receptor antagonist SU5416), and (3) in lungs from patients with idiopathic pulmonary arterial hypertension. Furthermore, we demonstrated that regulation of Fhl-1 was hypoxia-inducible transcription factor dependent. Abrogation of Fhl-1 expression in primary human pulmonary artery smooth muscle cells by small-interfering RNA suppressed, whereas Fhl-1 overexpression increased, migration and proliferation. Coimmunoprecipitation experiments identified Talin1 as a new interacting partner of Fhl-1. CONCLUSIONS: Protein screening identified Fhl-1 as a novel protein regulated in various forms of PH, including idiopathic pulmonary arterial hypertension.

Structure of pre-S2 N- and O-linked glycans in surface proteins from different genotypes of hepatitis B virus.

The middle-sized (M) surface proteins of hepatitis B virus (HBV) and other orthohepadnaviruses contain a conserved N-glycan in their pre-S2 domain, which is essential for the secretion of viral particles. Recently, we also found O-glycans in the pre-S2 domain of M protein from woodchuck hepatitis virus (WHV) and HBV genotype D. Since the O-glycosylation motif is not conserved in all genotypes of HBV, the glycosylation patterns of HBV genotypes A and C were analysed. Pre-S2 (glyco)peptides were released from HBV-carrier-derived HBV subviral particles by tryptic digestion, purified by reversed-phase HPLC and identified by amino acid and amino-terminal sequence analysis as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were characterized by anion-exchange chromatography, methylation analysis and on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS, demonstrating the presence of partially sialylated diantennary complex-type oligosaccharides in all genotypes examined. Pre-S2 O-glycans were characterized by on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS. The pre-S2 domain of M protein and, to a minor extent, of L (large) protein from HBV genotype C and D was partially O-glycosylated by Neu5Ac(alpha2-3)Gal(beta1-3)GalNAcalpha- or Gal(beta1-3)GalNAcalpha-units at Thr-37 within a conserved sequence context. Genotype A, containing no Thr at position 37 or 38, was not O-glycosylated. Analytical data further revealed that M protein is mostly amino-terminally acetylated in all examined genotypes and that the terminal methionine is partially oxidized. The findings may be relevant for the secretion and the immunogenicity of HBV.

The human Dnmt2 has residual DNA-(cytosine-C5) methyltransferase activity.

The human Dnmt2 protein is one member of a protein family conserved from Schizosaccharomyces pombe and Drosophila melanogaster to Mus musculus and Homo sapiens. It contains all of the amino acid motifs characteristic for DNA-(Cytosine-C5) methyltransferases, and its structure is very similar to prokaryotic DNA methyltransferases. Nevertheless, so far all attempts to detect catalytic activity of this protein have failed. We show here by two independent assay systems that the purified Dnmt2 protein has weak DNA methyltransferase activity. Methylation was observed at CG sites in a loose ttnCGga(g/a) consensus sequence, suggesting that Dnmt2 has a more specialized role than other mammalian DNA methyltransferases.