Structure of protease-cleaved escherichia coliα-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

Bacterial -2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli -2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli -2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli -2-macroglobulin and human -2-macro­globulin, this protease-activation mechanism is likely to operate across the diverse members of this group

The serum proteome of Atlantic salmon, Salmo salar, during pancreas disease (PD) following infection with salmonid alphavirus subtype 3 (SAV3)

Salmonid alphavirus is the aetological agent of pancreas disease (PD) in marine Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, with most outbreaks in Norway caused by SAV subtype 3 (SAV3). This atypical alphavirus is transmitted horizontally causing a significant economic impact on the aquaculture industry. This histopathological and proteomic study, using an established cohabitational experimental model, investigated the correlation between tissue damage during PD and a number of serum proteins associated with these pathologies in Atlantic salmon. The proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting. A number of humoral components of immunity which may act as biomarkers of the disease were also identified. For example, creatine kinase, enolase and malate dehydrogenase serum concentrations were shown to correlate with pathology during PD. In contrast, hemopexin, transferrin, and apolipoprotein, amongst others, altered during later stages of the disease and did not correlate with tissue pathologies. This approach has given new insight into not only PD but also fish disease as a whole, by characterisation of the protein response to infection, through pathological processes to tissue recovery. Biological significance: Salmonid alphavirus causes pancreas disease (PD) in Atlantic salmon, Salmo salar, and has a major economic impact on the aquaculture industry. A proteomic investigation of the change to the serum proteome during PD has been made with an established experimental model of the disease. Serum proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting with 72 protein spots being shown to alter significantly over the 12 week period of the infection. The concentrations of certain proteins in serum such as creatine kinase, enolase and malate dehydrogenase were shown to correlate with tissue pathology while other proteins such as hemopexin, transferrin, and apolipoprotein, altered in concentration during later stages of the disease and did not correlate with tissue pathologies. The protein response to infection may be used to monitor disease progression and enhance understanding of the pathology of PD

Crack initiation life estimations for notched specimens with residual stresses based on local strains

SIGLECopy held by FIZ Karlsruhe; available from UB/TIB Hannover / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

PS 07-14 vascular protein oxidation and redox proteomics in hypertension

Objective: Oxidative stress is implicated in hypertension (HTN) through redox-sensitive processes causing vascular damage. It remains unclear exactly how ROS cause vascular injury. We hypothesise that in HTN, increased ROS levels promote a shift of oxidative post-translational protein modifications from reversible into irreversible forms, leading to aberrant redox signalling and vascular injury. Design and Method: VSMCs from normotensive and hypertensive rats (WKY and SHRSP) were stimulated with AngII (10–7 M). Protein carbonylation was assessed by oxyblot. Protein tyrosine phosphatase (PTP)-oxidation was assessed by immunoblotting. Protein sulfenylation was detected using the DCP-Rho1 cell permeable, fluorescent probe. Differential gel electrophoresis (DiGE) and CyDye thiol labelling were employed for screening of reversibly oxidised proteome. Results: Irreversible protein carbonylation and PTP-hyperoxidation were increased in SHRSP compared to WKY (fold change (FC) = 1.29 and FC = 1.31, p < 0.05, respectively). AngII-stimulation induced PTP-hyperoxidation but not protein carbonylation in VSMCs from WKY rats (FC = 1.32 at 15 min, p < 0.05), with no alterations observed in SHRSP. On the contrary, reversible oxidation was reduced in SHRSP versus WKY, as demonstrated through thiol-proteome oxidation (13.6% (253 spots) decreased versus 6.7% (124 spots) increased oxidation), protein sulfenylation (FC = −1.78, p < 0.05) and PTP-oxidation (FC = −1.24, p < 0.05). AngII-stimulation tended to further decrease sulfenylation and PTP-oxidation levels in WKY. Proteomic data, filtered for FC > 2, detected 1777 spots with 377 (21%) being differentially oxidised between WKY and SHRSP. Candidate proteins exhibiting consistent changes across three replicates included β-actin (FC = 2.42), annexin A1 (−2.29), galectin-1 (1.83) and GAPDH (2.67). Conclusions: Our findings demonstrate that redox status in HTN is characterised by increased protein hyperoxidation and decreased levels of reversible oxidation. AngII is able to shift the balance between regulatory oxidation and hyperoxidation towards a hypertensive profile. Our findings identify differentially oxidised proteins in VSMCs in SHRSP vs WKY. These phenomena may be important in aberrant vascular signaling/function, contributing to oxidative vascular injury in HTN and associated target organ damage

41 Vascular protein oxidation and redox proteomics in human hypertension

Despite recent advances in the field of vascular redox signalling in hypertension, it still remains unclear exactly how ROS cause vascular injury. We hypothesise that regulation of redox-sensitive protein tyrosine phosphatases (PTP) through oxidative modification, is impaired in hypertension. VSMC normotensive (NT) and hypertensive (HT) individuals were stimulated with AngII (10-7 M) and ET-1 (10-7 M). Irreversible oxidation of proteins and PTPs was assessed by oxyblot. Differential gel electrophoresis (DiGE) and CyDye thiol labelling were employed for screening of reversibly oxidised proteome. Irreversible protein oxidation was not affected by AngII or ET-1 in VSMCs from NT and HT subjects. Proteomic data, filtered for FC >2, detected 2051 spots with 1899 (92.5%) being equally oxidised between NT and HT. In addition, oxidation of 57 (2.9%) spots was increased, while 95 (4.6%) were decreased in HT. Candidate proteins exhibiting consistent changes across three experimental replicates included β-actin (FC = –2.86), annexin A1 (–2.23), galectin-1 (–1.67), FK506 binding protein (–2.35) and polymerase I and transcript release factor (PTRF, –1.92). Stimulation with AngII altered the redox status in 2–3% of proteins, both in HT and NT. However, vimentin was the only target changing consistently across the replicates (FC = 2.48). Our findings indicate that pro-hypertensive agents may not impact significantly on irreversible protein and PTP oxidation in health and disease, but may have effects on reversible oxidation. Our proteomic data, in agreement with our previous rat studies, support decreased reversible thiol oxidation in hypertension

The plasma proteome and acute phase proteins of broiler chickens with gait abnormalities

The acute phase response (APR) is the early and non-specific systemic reaction of the innate immune system to homeostatic disturbances. Pro-inflammatory cytokines and chemokines released from macrophages, monocytes and infected and damaged tissues affect the synthesis and secretion of hepatocytic proteins and drastically alter the plasma protein profile. Plasma proteins that change concentrations as a result of an APR are termed acute phase proteins (APPs). The measurable changes in APP concentrations during infectious, inflammatory, stressful, traumatic or neoplastic events are often proportional to the severity of the event(s). As such APP measurements are used as disease biomarkers and for prognostication

Characterisation of the normal canine serum proteome using a novel electrophoretic technique combined with mass spectrometry

One dimensional (1D) serum protein electrophoresis (SPE) on agarose gels is a frequently used diagnostic tool for canine diseases; however, little is known regarding the precise composition of the different protein fractions in normal or diseased animals. In this study, to analyse the canine serum proteome in more detail, conventional 1D SPE was combined with second dimension (2D) polyacrylamide gel electrophoresis (PAGE), followed by tandem mass spectrometry (MS). One dimensional SPE was performed on the sera of 17 healthy dogs to establish normal reference ranges for the albumin and globulin sub-fractions. Two representative serum samples from healthy dogs were further separated using a novel method of 2D PAGE, leading to the generation of 26 distinct bands across the six main sub-fractions, which were subjected to MS analysis. Thirty-two proteins were identified, most of which were found in both dogs. Twenty proteins belonged specifically to the species Canis lupus familiaris, with the remaining 12 proteins belonging to other mammalian species, likely reflecting incomplete sequencing knowledge of canine proteins. Two dimensional electrophoresis and MS allowed identification of canine serum albumin precursor, serpin peptidase inhibitor, kininogen-1, vitamin D binding protein, haemopexin, complement C4 and a variety of immunoglobulin class molecules, along with localisation of these proteins within serum protein subfractions

Changes in the serum proteome of canine lymphoma identified by electrophoresis and mass spectrometry

The serum proteome of canine lymphoma was characterised by one dimensional (1D) serum protein electrophoresis (SPE) on agarose gels, two dimensional (2D) polyacrylamide gel electrophoresis (PAGE) and tandem mass spectrometry (MS). Results were compared with serum proteome data collected previously from the sera of healthy dogs. Twenty-one dogs with high grade multicentric lymphoma had significantly elevated quantities of α2 globulins on 1D SPE. Further separation of the serum proteins was performed on three dogs using a 2D PAGE system. Thirty-six different proteins were identified in 38 bands submitted for MS. Most of the proteins were the same as those previously identified in the sera of healthy dogs. Haptoglobin was identified in the sera of all three dogs with lymphoma and could account for the increased levels of α2 globulins. α2 Macroglobulin, α-antichymotrypsin and inter-α-trypsin inhibitor were also present in dogs with lymphoma. Clusterin, an anti-apoptotic protein, was identified in the serum of one dog with lymphoma. Kininogen, which is present in the sera of healthy dogs, was absent in all three dogs with lymphoma. The 2D electrophoresis technique identified alterations in the serum proteome of dogs with lymphoma and supported previous findings that canine lymphoma has an inflammatory component

Handling uncertainty in dynamic models: the pentose phosphate pathway in Trypanosoma brucei.

Dynamic models of metabolism can be useful in identifying potential drug targets, especially in unicellular organisms. A model of glycolysis in the causative agent of human African trypanosomiasis, Trypanosoma brucei, has already shown the utility of this approach. Here we add the pentose phosphate pathway (PPP) of T. brucei to the glycolytic model. The PPP is localized to both the cytosol and the glycosome and adding it to the glycolytic model without further adjustments leads to a draining of the essential bound-phosphate moiety within the glycosome. This phosphate “leak” must be resolved for the model to be a reasonable representation of parasite physiology. Two main types of theoretical solution to the problem could be identified: (i) including additional enzymatic reactions in the glycosome, or (ii) adding a mechanism to transfer bound phosphates between cytosol and glycosome. One example of the first type of solution would be the presence of a glycosomal ribokinase to regenerate ATP from ribose 5-phosphate and ADP. Experimental characterization of ribokinase in T. brucei showed that very low enzyme levels are sufficient for parasite survival, indicating that other mechanisms are required in controlling the phosphate leak. Examples of the second type would involve the presence of an ATP:ADP exchanger or recently described permeability pores in the glycosomal membrane, although the current absence of identified genes encoding such molecules impedes experimental testing by genetic manipulation. Confronted with this uncertainty, we present a modeling strategy that identifies robust predictions in the context of incomplete system characterization. We illustrate this strategy by exploring the mechanism underlying the essential function of one of the PPP enzymes, and validate it by confirming the model predictions experimentally