Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Enhancement of the viscosity of mucin by serum albumin.

The interaction of serum albumin with a model epithelial mucin from pig stomach was explored by rotary viscometry. During 30 min of incubation of human serum albumin(20mg/ml) and pig gastric mucin (8mg/ml) in iso-osmotic buffers at 37 degrees C, the solution became markedly viscous. Viscosity enhancement was proportional to albumin concentration (2-40mg/ml), was most pronounced under conditions of low shear rate (less than 45S-1), and was considerably greater than the additive or multiplicative viscosity values calculated from albumin or mucin solutions measured separately. The viscous mucin-albumin complex was destroyed by high shear rates (greater than 90S-1), but slowly re-formed under zero shear conditions. Elevation of pH (7 to 9), ionic strength (0.1 to 1.0), and addition of disodium EDTA (5mM) did not cause marked or specific alterations in the viscosity of the mixture, suggesting that electrostatic interactions probably do not stabilize mucin-albumin complexes. Urea (7M) and heating (35 to 55 degrees C) caused a major increase in the viscosity of mucin and mucin-albumin mixtures, suggesting that rupture of hydrogen bonds, unfolding and partial denaturation of mucin promotes greater intertangling (possibly hydrophobic interactions) between mucin and albumin molecules. The implications of mucin-albumin interaction in diseases associated with mucus obstruction are briefly discussed.     

Soft Tissue Artefacts of the Human Back: Comparison of the Sagittal Curvature of the Spine Measured Using Skin Markers and an Open Upright MRI

Soft tissue artefact affects the determination of skeletal kinematics. Thus, it is important to know the accuracy and limitations of kinematic parameters determined and modelled based on skin marker data. Here, the curvature angles, as well as the rotations of the lumbar and thoracic segments, of seven healthy subjects were determined in the sagittal plane using a skin marker set and compared to measurements taken in an open upright MRI scanner in order to understand the influence of soft tissue artefact at the back. The mean STA in the flexed compared to the extended positions were 10.2±6.1 mm (lumbar)/9.3±4.2 mm (thoracic) and 10.7±4.8 mm (lumbar)/9.2±4.9 mm (thoracic) respectively. A linear regression of the lumbar and thoracic curvatures between the marker-based measurements and MRI-based measurements resulted in coefficients of determination, R², of 0.552 and 0.385 respectively. Skin marker measurements therefore allow for the assessment of changes in the lumbar and thoracic curvature angles, but the absolute values suffer from uncertainty. Nevertheless, this marker set appears to be suitable for quantifying lumbar and thoracic spinal changes between quasi-static whole body postural changes.     

Über den Einfluss des Lichtes auf die Ablagerung von Pigment

Ohne Zusammenfassung     

Ubiquitin-specific protease 4 inhibits mono-ubiquitination of the master growth factor signaling kinase PDK1

Background

Phosphorylation by the phospho-inositide-dependent kinase 1 (PDK1) is essential for many growth factor-activated kinases and thus plays a critical role in various processes such as cell proliferation and metabolism. However, the mechanisms that control PDK1 have not been fully explored and this is of great importance as interfering with PDK1 signaling may be useful to treat diseases, including cancer and diabetes.

Methodology/Principal Findings

In human cells, few mono-ubiquitinated proteins have been described but in all cases this post-translational modification has a key regulatory function. Unexpectedly, we find that PDK1 is mono-ubiquitinated in a variety of human cell lines, indicating that PDK1 ubiquitination is a common and regulated process. Ubiquitination occurs in the kinase domain of PDK1 yet is independent of its kinase activity. By screening a library of ubiquitin proteases, we further identify the Ubiquitin-Specific Protease 4 (USP4) as an enzyme that removes ubiquitin from PDK1 in vivo and in vitro and co-localizes with PDK1 at the plasma membrane when the two proteins are overexpressed, indicating direct deubiquitination.

Conclusions

The regulated mono-ubiquitination of PDK1 provides an unanticipated layer of complexity in this central signaling network and offers potential novel avenues for drug discovery.     

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down‐regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.     

Lenalidomide Induces Lipid Raft Assembly to Enhance Erythropoietin Receptor Signaling in Myelodysplastic Syndrome Progenitors

Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.