Late events in the assembly of 20S proteasomes

Electron microscopy and STEM mass measurements have been used to characterize late intermediates in the assembly pathway of wildtype and mutant Rhodococcus proteasomes. A proteolytically inactive and processing-incompetent mutant, betaK33A, allowed a short-lived late intermediate of the pathway to be captured, the preholoproteasome. In this fully assembled 20S complex the 14 propeptides with an aggregate mass of 100 kDa fill the whole central cavity and most of the two antechambers. It is further shown that in wildtype Rhodococcus proteasomes the propeptides are degraded in a processive manner undergoing multiple cleavages before the products are discharged and the inner cavities are cleared. It appears that the docking of two half-proteasomes, i.e., preholoproteasome formation, is sufficient to trigger autocleavage of the Gly-1/Thr1 bond necessary for active site formation and the subsequent degradation of the propeptides.     

Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world’s leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1–893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/c mice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocular mucosa was well tolerated without signs of inflammation. N-PmpC-specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFNγ immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage.     

Escherichia coli ghosts promote innate immune responses in human keratinocytes

Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin.     

Structural coloration in a fossil feather

Investigation of feathers from the famous Middle Eocene Messel Oil Shale near Darmstadt, Germany shows that they are preserved as arrays of fossilized melanosomes, the surrounding beta-keratin having degraded. The majority of feathers are preserved as aligned rod-shaped eumelanosomes. In some, however, the barbules of the open pennaceous, distal portion of the feather vane are preserved as a continuous external layer of closely packed melanosomes enclosing loosely aligned melanosomes. This arrangement is similar to the single thin-film nanostructure that generates an iridescent, structurally coloured sheen on the surface of black feathers in many lineages of living birds. This is, to our knowledge, the first evidence of preservation of a colour-producing nanostructure in a fossil feather and confirms the potential for determining colour differences in ancient birds and other dinosaurs.     

New specimens of the avian taxa <Emphasis Type="Italic">Eurotrochilus</Emphasis> (Trochilidae) and <Emphasis Type="Italic">Palaeotodus</Emphasis> (Todidae) from the early Oligocene of Germany

We describe new specimens of stem group representatives of Trochilidae (hummingbirds) and Todidae (todies) from the Rupelian of Frauenweiler in southern Germany. The hummingbird fossil constitutes the fourth record of Eurotrochilus inexpectatus. It consists only of wing and pectoral girdle elements, but shows the previously unknown crista deltopectoralis of the humerus, whose shape differs from modern hummingbirds. The carpometacarpus bears a well-developed processus intermetacarpalis, which is a further synapomorphy of Eurotrochilus and crown group Trochilidae. The disarticulated partial skeleton of the tody allows a definitive taxonomic assignment of the Frauenweiler species to Palaeotodus itardiensis Mourer-Chauviré, and likewise exhibits so far unknown osteological details, including the morphologies of the quadrate and scapula. We further comment on the exceptional taphonomy and preservation of avian fossils from the Frauenweiler clay pit, where terrestrial birds are represented only by small to very small species, whose skeletons are always strongly disarticulated.     

Characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Beijing isolates from the Mediterranean area

Background  

The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases.     

Highlights from the 2010 BAS/BSCR spring meeting: New Frontiers in Cardiovascular Research

The British Atherosclerosis Society (BAS)/British Society for Cardiovascular Research (BSCR) spring meeting was held in Manchester, UK, on 7-8 June 2010. Experts in the field of systems biology, proteomics, metabolomics and miRNAs presented how these techniques can be used to discover 'New Frontiers in Cardiovascular Research'. The conference was attended by over 150 participants, mainly from the UK. A total of 2 days of presentations and a poster session with 55 posters provided the possibility to discuss the latest research results and showed the opportunities that new techniques can offer in cardiovascular research.     

Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution

Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.Myofilament proteins comprise the fundamental contractile apparatus of the heart, the cardiac sarcomere. They are subdivided into thin filament proteins, including actin, tropomyosin, the troponin complex (troponin C, troponin I, and troponin T), and thick filament proteins, including myosin heavy chains, myosin light chains, and myosin-binding protein C. Although calcium is the principal regulator of cardiac contraction through the excitation-contraction coupling process that culminates in calcium binding to troponin C, myofilament function is also significantly modulated by phosphorylation of constituent proteins, such as cardiac troponin I (cTnI),1 cardiac myosin-binding protein C (cMyBP-C), and myosin regulatory light chain (MLC-2). “Skinned” myocyte preparations from rodent hearts, in which the sarcolemmal envelope is disrupted through the use of detergents, have been invaluable in providing mechanistic information on the functional consequences of myofilament protein phosphorylation following exposure to neurohormonal stimuli that activate pertinent kinases prior to skinning or direct exposure to such kinases in active form after skinning (for recent examples, see studies on the phosphorylation of cTnI (1–3), cMyBP-C (4–6), and MLC-2 (7–9)). Nevertheless, to date, only a few myofilament proteins have been studied using proteomics (10–19), and a detailed proteomic characterization of the myofilament subproteome and its associated proteins from skinned myocytes has not been performed. In the present analysis, we used an LTQ Orbitrap XL equipped with ETD (20) to analyze the subproteome of skinned cardiomyocytes with or without prior stimulation. Endothelin-1 and isoproterenol were used to activate the endothelin receptor/protein kinase C and β-adrenoreceptor/protein kinase A pathway, respectively (21, 22). Importantly, the mass accuracy of the Orbitrap mass analyzer helped to distinguish true phosphorylation sites from false assignments, and the sensitivity of the ion trap provided novel insights into the translocation of phosphatase regulatory and targeting subunits following β-adrenergic stimulation.     

Proteomics Characterization of Extracellular Space Components in the Human Aorta

The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.Vascular cells, in particular vascular smooth muscle cells, produce and maintain a complex meshwork of ECM.¹ The ECM is not only the scaffold for the anchorage and mobility of residing cells but also absorbs and transduces the shear and strain forces of the blood flow. It is primarily composed of elastin, collagen, proteoglycans, and glycoproteins. The elastin fibers and type I and III fibrillar collagens form a rigid network of highly cross-linked interstitial matrix. They offer elasticity (elastin) and tensile strength (collagens). Proteoglycans, because of their negative charge, attract water and confer resistance to compression. Finally, glycoproteins participate in matrix organization and are essential for cell attachment.The vascular ECM also serves as a substrate for the binding and retention of secreted, soluble proteins of vascular cells as well as molecules coming from the circulation, including lipoproteins, growth factors, cytokines, proteases, and protease inhibitors. These components are invariably associated with ECM proteins, especially proteoglycans. Together they comprise the vascular extracellular environment and are pivotal for disease processes, such as atherosclerosis and aneurysm formation (1).Although proteomics has been previously applied to vascular tissues, only one study has specifically targeted the extracellular vascular environment (2). This study was focused on the isolation of intimal proteoglycans from human carotid arteries. Moreover, most proteomics studies use whole tissue lysates, which are rich in cellular proteins that inevitably mask the identification of the less abundant proteins of the vascular extracellular environment (3–5). Thus, the composition of the vascular ECM and its associated proteins remains poorly defined. In the present study, we used morphologically normal human aortic samples to develop a method for the extraction of proteins present in the extracellular environment, including ECM proteins and proteins attached to the ECM. We had three specific aims: first, to reduce the contamination with cellular proteins, thereby increasing the chance of identifying scarce extracellular proteins; second, to efficiently solubilize and deglycosylate ECM proteins to improve their analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS); and third, to interface the nanoflow LC system to a recently developed injection device, which splits the flow from the analytical column, to allow the reanalysis of the same sample during a single LC-MS/MS run (RePlay, Advion).Our methodology provides a detailed overview of the aortic ECM and its associated proteins, many reported for the first time in proteomics analysis of the vasculature. Most importantly, this method could be adapted for use with other tissues to further our understanding of the composition of extracellular environment and ECM turnover under various disease conditions.     

Stabilisation of human interferon β synthesis in Escherichia coli by zinc ions

Human interferon β synthesized in Escherichia coli is unstable and toxic for the bacterial cell. Zinc ions are able to stabilise interferon β in E. coli probably by inhibiting the action of cell internal proteinase(s) which affect the half-life of this foreign protein. As a result up to one order of magnitude more active IFN-β can be detected in Zn²⁺-treated E. coli cells.