Convenient synthesis of sulfated oligofucosides

Two types of sulfated octyl tetra- to octaoligofucosides with different sulfation patterns were synthesized employing a combination of stepwise elongation and convergent strategies in which trichloroacetimidates and thioglycosides were selected as the glycosyl donors.     

SLRP interaction can protect collagen fibrils from cleavage by collagenases

Decorin, fibromodulin and lumican are small leucine-rich repeat proteoglycans (SLRPs) which interact with the surface of collagen fibrils. Together with other molecules they form a coat on the fibril surface which could impede the access to collagenolytic proteinases. To address this hypothesis, fibrils of type I or type II collagen were formed in vitro and treated with either collagenase-1 (MMP1) or collagenase-3 (MMP13). The fibrils were either treated directly or following incubation in the presence of the recombinant SLRPs. The susceptibility of the uncoated and SLRP-coated fibrils to collagenase cleavage was assessed by SDS/PAGE. Interaction with either recombinant decorin, fibromodulin or lumican results in decreased collagenase cleavage of both fibril types. Thus SLRP interaction can help protect collagen fibrils from cleavage by collagenases.     

RACK1 recruits STAT3 specifically to insulin and insulin-like growth factor 1 receptors for activation, which is important for regulating anchorage-independent growth

Current understanding of the activation of STATs is through binding between the SH2 domain of STATs and phosphotyrosine of tyrosine kinases. Here we demonstrate a novel role of RACK1 as an adaptor for insulin and insulin-like growth factor 1 receptor (IGF-1R)-mediated STAT3 activation specifically. Intracellular association of RACK1 via its N-terminal WD domains 1 to 4 (WD1-4) with insulin receptor (IR)/IGF-1R is augmented upon respective ligand stimulation, whereas association with STAT3 is constitutive. Purified RACK1 or RACK1 WD1-4 associates directly with purified IR, IGF-1R, and STAT3 in vitro. Insulin induces multiprotein complex formation of RACK1, IR, and STAT3. Overexpression or downregulation of RACK1 greatly enhances or decreases, respectively, IR/IGF-1R-mediated activation of STAT3 and its target gene expression. Site-specific mutants of IR and IGF-1R impaired in RACK1 binding are ineffective in mediating recruitment and activation of STAT3 as well as in insulin- or IGF-1-induced protection of cells from anoikis. RACK1-mediated STAT3 activation is important for insulin and IGF-1-induced anchorage-independent growth in certain ovarian cancer cells. We conclude that RACK1 mediates recruitment of STAT3 to IR and IGF-1R specifically for activation, suggesting a general paradigm for the need of an adaptor in mediating activation of STATs by receptor protein tyrosine kinases.     

A SERS-based immunoassay with highly increased sensitivity using gold/silver core-shell nanorods

An integrated platform for a very sensitive detection of cocaine based on a refractometric biosensor is demonstrated. The system uses a waveguide grating biosensor functionalized with a cocaine multivalent antigen-carrier protein conjugate. The immunoassay scheme consists of the competitive binding of cocaine-specific antibodies to the immobilized conjugates. A 1000-fold enhancement of the sensor's sensitivity is achieved when using gold conjugated monoclonal antibodies instead of free antibodies. Together with the optimization of the assay conditions, the setup is designed for a quick identification of narcotics using automated sampling. The results show that the presence of cocaine in a liquid sample could be identified down to a concentration of 0.7 nM within one minute. This value can be reduced even further when longer binding time is allowed (0.2 nM after 15 min). Application of the system to detection of narcotics at airport security control points is discussed.     

Resveratrol inhibits inflammatory responses via the mammalian target of rapamycin signaling pathway in cultured LPS-stimulated microglial cells

Background

Resveratrol have been known to possess many pharmacological properties including antioxidant, cardioprotective and anticancer effects. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, the precise mechanisms for its beneficial effects are still not fully understood. We investigate the effect of anti-inflammatory and mechamisms of resveratrol by using lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells.

Methodology/Principal Findings

BV-2 cells were treated with resveratrol (25, 50, and 100 µM) and/or LPS (1 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10), Akt, mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) cascades, inhibitor κB-α (IκB-α) and cyclic AMP-responsive element-binding protein (CREB) were measured by western blot. Resveratrol significantly attenuated the LPS-induced expression of NO, PGE2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor-κB (NF-κB) in BV-2 cells. Resveratrol increased PTEN, Akt and mTOR phosphorylation in a dose-dependent manner or a time-dependent manner. Rapamycin (10 nM), a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced microglial activation. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IκB-α, CREB, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK).

Conclusion and Implications

This study indicates that resveratrol inhibited LPS-induced proinflammatory enzymes and proinflammatory cytokines via down-regulation phosphorylation of NF-κB, CREB and MAPKs family in a mTOR-dependent manner. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of resveratrol.     

An MRM-based workflow for quantifying cardiac mitochondrial protein phosphorylation in murine and human tissue

The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics.     

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water (²H₂O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with ²H₂O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein half-life to be determined without plateau-level ²H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d⁻¹ and 0.163 d⁻¹, respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (∼60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.Mitochondrial dysfunctions are observed in disorders such as neurodegeneration, cardiovascular diseases, and aging (1–3). It is postulated that the failure to contain or replenish mitochondrial proteins damaged by reactive oxygen species directly underlies many pathological phenotypes (4). The development of effective treatments for these diseases therefore relies on understanding the molecular basis of protein dynamics. Outstanding questions are how the processes of mitochondrial proteome dynamics are regulated in different systems, and how their perturbations could progress to pathological remodeling of the organelle. Thus far, quantitative proteomics efforts have been predominated by steady-state measurements, which often provide fragmentary snapshots of the proteome that are difficult to comprehend in the context of other cellular events.To further understand mitochondrial dynamics in vivo, we examined the turnover rates of individual heart and liver mitochondrial proteins on a proteome scale. Both the liver and the heart contain large numbers of mitochondria, but cardiac and hepatic mitochondria differ in their protein composition, oxygen consumption, substrate utilization, and disease manifestation. However, these differences are often interpreted only by protein compositions and steady-state abundance, without the consideration of protein kinetics in the temporal dimension. Abnormal protein kinetics may indicate dysfunctions in protein quality control, the accumulation of damaged proteins, misfolding, or other proteinopathies. Protein dynamics itself is an important intrinsic property of the proteome, the disruption of which could be causal of cellular etiologies.At minimum, a kinetic definition of the proteome requires knowledge of the rate at which individual proteins are being replaced. Isotope tracers are particularly useful for tracking such continual renewal of the proteome in living systems, because they allow differentiation between preexisting and newly synthesized proteins (5). Among the available stable isotope precursors, heavy water (²H₂O) labeling offers several advantages with respect to safety, labeling kinetics, and cost (6, 7). First, ²H₂O administration to animals and humans at low enrichment levels is safe for months or even years (8). Second, maintaining constant ²H enrichment levels in body water following the initial intake of ²H₂O is easily achieved, because administrated ²H₂O rapidly equilibrates over all tissues but decays slowly (9, 10). Third, ²H₂O labeling is more cost effective than other stable isotope labeling methods. Importantly, ²H₂O intake induces universal ²H incorporation into biomolecules. Systematic insights into protein turnover in vivo could therefore be correlated to that of nucleic acids, carbohydrates, or lipids, enabling broad applications for this technology in studying mammalian systems, including humans.A variety of methodologies have been developed to analyze the extent of ²H incorporation in proteins following ²H₂O labeling, including GC-MS measurements of hydrolyzed target proteins (11–14) and peptide analysis in MALDI-TOF MS (15) and LC-MS (16, 17). More recently, Price et al. described an approach for measuring protein turnover by calculating the theoretical number of ²H-labeling sites on a peptide sequence (18) and reported the turnover rates of ∼100 human plasma proteins. Here we describe another novel strategy to determine protein turnover rates on a proteomic scale using ²H₂O labeling. By computing the parameters needed to deduce fractional protein synthesis using software we developed, we were able to obtain protein half-life data without relying on the asymptotic isotopic abundance of peptide ions. Our approach also has the unique benefit of automating all steps of isotopomer quantification and postcollection data analysis, and it does not require knowledge of the exact precursor enrichment or labeling sites of peptides. We observed diverse kinetics from 458 liver and heart mitochondrial proteins that inform essential characteristics of mitochondrial dynamics and intragenomic differences between the two organs.     

A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.