Pronova Biopharma - Vurdering av utslipp til Sandefjordsfjorden

Det er gjort en vurdering av hvordan avløpsvann fra Pronova Biopharma kan påvirke oksygenforholdene i Sandefjordsfjorden. Arbeidet har bl.a. omfattet strømmålinger, oksygenprøver og bruk av modeller. Hovedkonklusjonen er at til vanlig vil utslippet bare i meget liten grad påvirke oksygenforholdene. De viktigste elementene i fjordens oksygenbudsjett varierer med tiden, og høsten er den mest sårbare perioden. Man kan ikke se helt bort fra at et sammentreff av flere ugunstige faktorer da kan skape en periode med en merkbar redusert konsentrasjon (til vannkvalitetsklasse Mindre God) i en lokal vannmasse omkring utslippet. Ingen undersøkelser har imidlertid påvist at dette har skjedd.Pronova Biopharma AS, Sandefjord v/ A.L. Steneru

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32?36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component

Type B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins

Molecular events that render pancreatic beta cells targets of T lymphocytes

© 2012 Dr. Dhana Govind GorasiaType 1 Diabetes (T1D), which affects approximately 140000 people in Australia, is an autoimmune disease that results from specific destruction of insulin-producing pancreatic beta cells. Individuals suffering from T1D have to take exogenous insulin to survive. Studies using a non obese diabetic (NOD) mouse model have revealed that multiple immune cells, such as macrophages, B cells and T lymphocytes infiltrate the islets and destroy beta cells via different effector mechanisms. One group of identified mediators of beta cell death is pro-inflammatory cytokines, such as IFN-γ, TNF-α and IL-1β, which are produced by the infiltrating immune cells. In this study, the effects of a combination of pro-inflammatory cytokine treatments on beta (NIT-1) and alpha (αTC-1) cell lines as well as isolated mouse islets were assessed using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). A total of 58 proteins in NIT-1 cells and 55 in αTC-1 cells were differentially expressed when exposed to different combinations of cytokines. Protein identification by mass spectrometry (MS) revealed that the major difference in the protein expression between cytokine treated NIT-1 and αTC-1 cells were stress induced proteins and free radical scavenging enzymes. Stress induced phosphoprotein 1 and heat shock proteins were observed to be altered in cytokine treated NIT-1 cells and free radical scavenging enzymes such as superoxide dismutase 2 (SOD2) and peroxiredoxin 3 were observed to be upregulated in cytokine treated αTC-1 cells. These data are indicative of NIT-1 cells exhibiting cellular stress upon cytokine treatment, reflecting insufficient defense against oxidative stress. In contrast, αTC-1 cells were able to scavenge free radicals hence did not exhibit cellular stress under the same conditions. To investigate if these pathways were also important in cytokine responses of purified islets, the effect of IFN-γ, IL-1β and TNFα on protein expression of islets was examined. Subcellular fractionation was performed to increase the coverage of the islet proteome. Treatment of islets with cytokines caused an expression change in 254, 236 and 154 proteins in the cytosol, membrane/organelle and nuclear fraction, respectively. Protein identification by MS showed that the expression levels of heat shock proteins were altered and also SOD2 levels were upregulated. Peroxiredoxin levels remained unaltered indicating an ineffective defense response against free radicals and subsequent cellular stress within islets. Proteomics interrogation of cytokine treated beta cells also revealed changes in the levels of Endoplasmic Reticulum (ER) foldases which may affect the folding of proinsulin in the ER. In this study, NIT-1 cells expressing Flag tagged proinsulin were generated in order to determine which foldases interact with proinsulin, and therefore play a role in proinsulin folding. Co-immunoprecipitation studies revealed that BiP, DnajB11 and PDIA6 together with other ER foldases associated with proinsulin. PDIA6 had not previously been associated with disulfide bond formation in proinsulin. The foldases that interact with misfolded proinsulin were also explored. NIT-1 cells expressing Flag tagged proinsulin with the Akita mutation (C96Y) were generated. Co-immunoprecipitation studies revealed that the majority of chaperones that interacted with wild type proinsulin also interacted with misfolded Akita proinsulin except for P58IPK which only interacted with misfolded proinsulin. Interestingly, more PDIA6 was associated with the misfolded form compared to wild type proinsulin. These results indicate a possible involvement of PDIA6 in the reduction of disulfide bonds in misfolded proinsulin, thereby enabling misfolded proinsulin to enter the ER associated degradation (ERAD) pathway. Pro-inflammatory cytokines are also known to cause an upregulation of MHC class I molecules on the beta cell surface. IFN-γ causes an upregulation of tapasin levels, which is a chaperone involved in MHC class I synthesis and is known to play a role in the editing of peptides presented by MHC class I molecules. In this study, the effect of tapasin upregulation on antigen processing and presentation in the absence of cytokines was examined. NIT-1 cells overexpressing tapasin were generated. Tapasin upregulation had no effect on the expression of MHC class I molecules on the cell surface. Peptides presented by MHC class I molecules were analysed in both NIT-1 and NIT-1 cells overexpressing tapasin. No changes were observed in the sequences of peptides presented on MHC class I molecules in the presence of elevated tapasin. Nevertheless, NIT-1 peptidome was established. Thus tapasin on its own was shown not to have a profound effect on the type of antigen presented on MHC class I molecules

The Type IX Secretion System: Advances in Structure, Function and Organisation

The type IX secretion system (T9SS) is specific to the Bacteroidetes phylum. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilises the T9SS to transport many proteins—including its gingipain virulence factors—across the outer membrane and attach them to the cell surface. Additionally, the T9SS is also required for gliding motility in motile organisms, such as Flavobacterium johnsoniae. At least nineteen proteins have been identified as components of the T9SS, including the three transcription regulators, PorX, PorY and SigP. Although the components are known, the overall organisation and the molecular mechanism of how the T9SS operates is largely unknown. This review focusses on the recent advances made in the structure, function, and organisation of the T9SS machinery to provide further insight into this highly novel secretion system

Protein Interactome Analysis of the Type IX Secretion System Identifies PorW as the Missing Link between the PorK/N Ring Complex and the Sov Translocon

International audienceThe type IX secretion system (T9SS) transports cargo proteins through the outer membrane of Bacteroidetes and attaches them to the cell surface for func- tions including pathogenesis, gliding motility, and degradation of carbon sources. The T9SS comprises at least 20 different proteins and includes several modules: the trans- envelope core module comprising the PorL/M motor and the PorK/N ring, the outer membrane Sov translocon, and the cell attachment complex. However, the spatial or- ganization of these modules is unknown. We have characterized the protein interac- tome of the Sov translocon in Porphyromonas gingivalis and identified Sov-PorV-PorA as well as Sov-PorW-PorN-PorK to be novel networks. PorW also interacted with PGN_1783 (PorD), which was required for maximum secretion efficiency. The identifi- cation of PorW as the missing link completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore

Protein Interactome Analysis of the Type IX Secretion System Identifies PorW as the Missing Link between the PorK/N Ring Complex and the Sov Translocon

International audienceThe type IX secretion system (T9SS) transports cargo proteins through the outer membrane of Bacteroidetes and attaches them to the cell surface for functions including pathogenesis, gliding motility, and degradation of carbon sources. The T9SS comprises at least 20 different proteins and includes several modules: the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, the outer membrane Sov translocon, and the cell attachment complex. However, the spatial organization of these modules is unknown. We have characterized the protein interactome of the Sov translocon in Porphyromonas gingivalis and identified Sov-PorV-PorA as well as Sov-PorW-PorN-PorK to be novel networks. PorW also interacted with PGN_1783 (PorD), which was required for maximum secretion efficiency. The identification of PorW as the missing link completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore. IMPORTANCE The T9SS is a newly identified protein secretion system of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum used by pathogens associated with diseases of humans, fish, and poultry for the secretion and cell surface attachment of virulence factors. The T9SS comprises three known modules: (i) the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, (ii) the outer membrane Sov translocon, and (iii) the cell surface attachment complex. The spatial organization and interaction of these modules have been a mystery. Here, we describe the protein interactome of the Sov translocon in the human pathogen Porphyromonas gingivalis and have identified PorW as the missing link which bridges PorN with Sov and so completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing, for the first time, a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore

Protein Interactome Analysis of the Type IX Secretion System Identifies PorW as the Missing Link between the PorK/N Ring Complex and the Sov Translocon

International audienceThe type IX secretion system (T9SS) transports cargo proteins through the outer membrane of Bacteroidetes and attaches them to the cell surface for func- tions including pathogenesis, gliding motility, and degradation of carbon sources. The T9SS comprises at least 20 different proteins and includes several modules: the trans- envelope core module comprising the PorL/M motor and the PorK/N ring, the outer membrane Sov translocon, and the cell attachment complex. However, the spatial or- ganization of these modules is unknown. We have characterized the protein interac- tome of the Sov translocon in Porphyromonas gingivalis and identified Sov-PorV-PorA as well as Sov-PorW-PorN-PorK to be novel networks. PorW also interacted with PGN_1783 (PorD), which was required for maximum secretion efficiency. The identifi- cation of PorW as the missing link completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore

Protein substrates of a novel secretion system are numerous in the bacteroidetes phylum and have in common a cleavable C-Terminal secretion signal, extensive post-translational modification, and cell-surface attachment

The research outputs in this collection have been funded in whole or in part by the National Health and Medical Research Council (NHMRC).Published VersionCopyright © 2012 American Chemical SocietyThe secretion of certain proteins in Porphyromonas gingivalis is dependent on a C-terminal domain (CTD). After secretion, the CTD is cleaved prior to extensive modification of the mature protein, probably with lipopolysaccharide, therefore enabling attachment to the cell surface. In this study, bioinformatic analyses of the CTD demonstrated the presence of three conserved sequence motifs. These motifs were used to construct Hidden Markov Models (HMMs) that predicted 663 CTD-containing proteins in 21 fully sequenced species of the Bacteroidetes phylum, while no CTD-containing proteins were predicted in species outside this phylum. Further HMM searching of Cytophaga hutchinsonii led to a total of 171 predicted CTD proteins in that organism alone. Proteomic analyses of membrane fractions and culture fluid derived from P. gingivalis and four other species containing predicted CTDs (Parabacteroides distasonis, Prevotella intermedia, Tannerella forsythia, and C. hutchinsonii) demonstrated that membrane localization, extensive post-translational modification, and CTD-cleavage were conserved features of the secretion system. The CTD cleavage site of 10 different proteins from 3 different species was determined and found to be similar to the cleavage site previously determined in P. gingivalis, suggesting that homologues of the C-terminal signal peptidase (PG0026) are responsible for the cleavage in these species.10.1021/pr400487

Type B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins