Water soluble nanoparticles from PEG-based cationic hyperbranched polymer and RNA that protect RNA from enzymatic degradation

Recent advances in understanding biological systems have proven that RNA is not merely the carrier of genetic information, but also a key molecule in regulation of gene expression and other crucial metabolic processes. Therefore, it is being considered as an ideal therapeutic candidate both for metabolic and genetic disorders. However, research involving RNA molecules faces a practical limitation since RNA is highly labile. We have developed a novel method to protect RNA from cleavage by complexing it with a hyperbranched cationic polymer. It was found that total cellular RNA isolated from yeast spontaneously interacts with the positively charged polymer to form a spherical nanoparticle morphology. This interaction protects the RNA against enzymatic degradation. This methodology can be easily adapted for long-term storage of RNA, long distance transfer of RNA, and genetic engineering using RNA as a building block.     

Deletion of Cysteine Cathepsins B or L Yields Differential Impacts on Murine Skin Proteome and Degradome

Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb^−/−, and Ctsl^−/− mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl^−/− skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl^−/− skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl^−/− or Ctsb^−/− samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.Cathepsins B and L are ubiquitously expressed papain-like cysteine proteases belonging to the C1a papain family (clan CA), with 11 members in humans (1) and 18 members in mice (2). Most cysteine cathepsins like cathepsin L are endopeptidases, whereas cathepsin B shows both endopeptidase and carboxydipeptidase activity (3). Mainly localized in the endosomal/lysosomal compartment, cathepsins have traditionally been thought to play important roles in lysosomal protein turnover. Additional specific functions have been postulated that link cathepsins to different physiological and pathological processes.Studies using cathepsin L (Ctsl)-gene-deficient mice¹ revealed an important role of Ctsl in cardiac homeostasis (4–6) and a contribution of Ctsl to MHC II-mediated antigen presentation (7, 8) and prohormone processing (9, 10). In a mouse model of pancreatic neuroendocrine cancer, Ctsl promoted tumor growth and invasiveness (11, 12). In stark contrast, Ctsl was found to attenuate tumor progression in mouse models of skin cancer, highlighting the context-specific function of this protease (13, 14).The most prominent phenotype of Ctsl-deficient mice is periodic hair loss together with epidermal hyperplasia, acanthosis, and hyperkeratosis (15). These alterations in skin morphology are assumed to be keratinocyte specific, as controlled re-expression of Ctsl under a keratin 14 promoter results in inconspicuous epidermal proliferation (16). The hair loss phenotype is caused by increased apoptosis and proliferation of hair follicle keratinocytes during the regression phase (catagen) of the hair follicle (17).Cathepsin B (Ctsb)-gene-deficient mice do not display a spontaneous phenotype (18, 19), but if pathologically challenged these mice are less susceptible to disease in pancreatitis (20) and are less affected by TNFα-induced hepatocyte apoptosis (21). In tumor models of metastasizing breast cancer and pancreatic neuroendocrine neoplasias, mice deficient in Ctsb showed delayed cancer progression and reduced invasion (11, 22, 23). As good corroboration, the overexpression of Ctsb in the mouse mammary tumor virus–polyoma middle T breast cancer model promotes a more severe tumor phenotype (24).In contrast to single-gene-deficient mice, mice with a double deficiency in both Ctsb and Ctsl die 4 weeks after birth as a result of pronounced lysosomal storage disease leading to neuron death in the cerebral cortex and the degeneration of cerebellar Purkinje cells (25). Because single-gene-deficient mice do not show autophagolysosomal and lysosomal accumulations in neurons, mutual compensation between Ctsb and Ctsl in vivo has been suggested (26).The present proteomic study focuses on the molecular roles of Ctsb and Ctsl in skin homeostasis. We applied a 2-fold strategy consisting firstly of a gel-free quantitative proteomic approach (27, 28) to investigate protein alterations. Secondly, we performed terminal amine isotopic labeling of substrates (TAILS) (29) to determine changes in the skin proteome cleavage pattern and to identify Ctsb- and Ctsl-dependent cleavage events. Selected proteomic data were corroborated by means of immunodetection and immunohistochemistry. Selected mRNA levels were determined via qPCR in order to discriminate expression changes from posttranslational alterations. We identified specific proteomic and degradomic effects stemming from the deletion of either Ctsb or Ctsl. Our findings highlight the pivotal function of these proteases in maintaining proteome homeostasis and in balancing the proteolytic network. This is one of the first studies investigating how the deletion of individual proteases affects proteolytic processing in vivo.     

BRCT domains: Easy as one,two, three

BRCA1 C-terminal (BRCT) domains are integral signaling modules in the DNA damage response (DDR). Aside from their established roles as phospho-peptide binding modules, BRCT domains have been implicated in phosphorylation-independent protein interactions, DNA binding and poly(ADP-ribose) (PAR) binding. These numerous functions can be attributed to the diversity in BRCT domain structure and architecture, where domains can exist as isolated single domains or assemble into higher order homo- or hetero-domain complexes. In this review, we incorporate recent structural and biochemical studies to demonstrate how structural features allow single and tandem BRCT domains to attain a high degree of functional diversity.Key words: BRCT domain, DNA repair, phosphorylation, phospho-peptide interaction, protein interaction, DNA binding, DNA damage response     

Synthesis and characterization of carboxylic acid conjugated, hydrophobically derivatized, hyperbranched polyglycerols as nanoparticulate drug carriers for cisplatin

Hyperbranched polyglycerols (HPGs) with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) were synthesized and further functionalized with carboxylate groups to bind and deliver cisplatin. Low and high levels of carboxylate were conjugated to HPGs (HPG-C(8/10)-MePEG(6.5)-COOH(113) and HPG-C(8/10)-MePEG(6.5)-COOH(348)) and their structures were confirmed through NMR and FTIR spectroscopy and potentiometric titration. The hydrodynamic diameter of the HPGs ranged from 5-10 nm and the addition of COOH groups decreased the zeta potential of the polymers. HPG-C(8/10)-MePEG(6.5)-COOH(113) bound up to 10% w/w cisplatin, whereas HPG-C(8/10)-MePEG(6.5)-COOH(348) bound up to 20% w/w drug with 100% efficiency. Drug was released from HPG-C(8/10)-MePEG(6.5)-COOH(113) over 7 days at the same rate, regardless of the pH. Cisplatin release from HPG-C(8/10)-MePEG(6.5)-COOH(348) was significantly slower than HPG-C(8/10)-MePEG(6.5)-COOH(113) at pH 6 and 7.4, but similar at pH 4.5. Release of cisplatin into artificial urine was considerably faster than into buffer. Carboxylated HPGs demonstrated good biocompatibility, and drug-loaded HPGs effectively inhibited proliferation of KU-7-luc bladder cancer cells.     

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.     

Conjugation to hyperbranched polyglycerols improves RGD-mediated inhibition of platelet function in vitro

RGD (arginine-glycine-aspartic acid) is a known peptide sequence that binds platelet integrin GPIIbIIIa and disrupts platelet-fibrinogen binding and platelet cross-linking during thrombosis. RGD peptides are unsuitable for clinical applications due to their high 50% inhibitory concentration (IC50) and low in vivo residence times. We addressed these issues by conjugating RGD peptides to biocompatible macromolecular carriers: hyperbranched polyglycerols (HPG) via divinyl sulfone. The GPIIbIIIa binding activity of RGD was maintained after conjugation and the effectiveness of the HPG-RGD conjugate was dependent upon molecular weight and the number of RGD peptides attached to each HPG molecule. These polyvalent inhibitors of platelet aggregation decreased the IC50 of RGD in an inverse linear manner based on the number of RGD peptides per HPG. Since HPG-RGD conjugates do not cause platelet activation by degranulation and certain substitution ratios do not increase fibrinogen binding to resting platelets, HPG-RGD may serve as a model for a novel class of antithrombotics.     

The influence of grafted polymer architecture and fluid hydrodynamics on protein separation by entropic interaction chromatography

Entropic interaction chromatography (EIC) provides efficient size-based separation of protein mixtures through the entropy change associated with solute partitioning into a layer of hydrophilic homopolymer that has been end-grafted within the pores of a macroporous chromatography support. In this work, surface-initiated atom-transfer radical polymerization (ATRP) is used to prepare a library of EIC stationary phases covering a wide range of grafted-chain densities and molecular weights. Exhaustive chain cleavage and analysis by saponification and GPC-MALLS, respectively, show that the new ATRP synthesis procedure allows for excellent control over graft molecular weight and polydispersity. The method is used to prepare high-density grafts (up to 0.164 +/- 0.005 chains/nm(2)) that extend the range of EIC applications to include efficient buffer-exchange and desalting of protein preparations. Reducing the graft density allows for greater partitioning of high molecular weight solutes, extending the linear range of the selectivity curve. Increasing graft molecular weight also alters selectivity, but more directly affects column capacity by increasing the volume of the grafted layer. Protein partitioning in high-density EIC columns is found to decrease with mobile-phase velocity (u). Although solute mass transfer resistances leading to an increase in plate height can explain this effect, pressure drop data across the column are indicative of weak convective flow through at least a fraction of the grafted architecture. Modeling of the grafted brush properties in the presence of solvent flow by subjecting a self-consistent-field theory representation of the brush to a viscous shear force predicts that the grafted chains will tilt and elongate in the direction of flow. The shear force may therefore act to reduce the number of conformations available to chains, increasing their rigidity without significantly altering the thickness of the grafted layer. A reduction in protein partitioning is then predicted when the dependence on u of the solute entropy loss is stronger than that of the grafted polymer, a condition met at high graft densities.     

Metabolic footprint of epiphytic bacteria on Arabidopsis thaliana leaves

The phyllosphere, which is defined as the parts of terrestrial plants above the ground, is a large habitat for different microorganisms that show a high extent of adaption to their environment. A number of hypotheses were generated by culture-independent functional genomics studies to explain the competitiveness of specialized bacteria in the phyllosphere. In contrast, in situ data at the metabolome level as a function of bacterial colonization are lacking. Here, we aimed to obtain new insights into the metabolic interplay between host and epiphytes upon colonization of Arabidopsis thaliana leaves in a controlled laboratory setting using environmental metabolomics approaches. Quantitative nuclear magnetic resonance (NMR) and imaging high-resolution mass spectrometry (IMS) methods were used to identify Arabidopsis leaf surface compounds and their possible involvement in the epiphytic lifestyle by relative changes in compound pools. The dominant carbohydrates on the leaf surfaces were sucrose, fructose and glucose. These sugars were significantly and specifically altered after epiphytic leaf colonization by the organoheterotroph Sphingomonas melonis or the phytopathogen Pseudomonas syringae pv. tomato, but only to a minor extent by the methylotroph Methylobacterium extorquens. In addition to carbohydrates, IMS revealed surprising alterations in arginine metabolism and phytoalexin biosynthesis that were dependent on the presence of bacteria, which might reflect the consequences of bacterial activity and the recognition of not only pathogens but also commensals by the plant. These results highlight the power of environmental metabolomics to aid in elucidating the molecular basis underlying plant–epiphyte interactions in situ.     

The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis

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Highlights

• •Mitochondrial heart N terminome shows aminopeptidase processing after MTS cleavage.
• •CLPP-deficiency alters protein processing patterns in mouse heart mitochondria.
• •Candidate substrates identified by N termini accumulation and interaction with inactive ClpXP.
• •UQCRC1, HSPA9 and OAT validated biochemically as high confidence ClpXP substrates.

     

Hyperbranched Polyglycerol as a Colloid in Cold Organ Preservation Solutions

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.