Genetic, structural, and molecular insights into the function of ras of complex proteins domains

Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural informa- tion available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.

Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation

In common with other positive-strand RNA viruses, replication of feline calicivirus (FCV) results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. In this study, bioinformatics approaches have identified three of the FCV non-structural proteins, namely p32, p39 and p30, as potential transmembrane proteins. These proteins were able to target enhanced cyan fluorescent protein to membrane fractions where they behaved as integral membrane proteins. Immunofluorescence microscopy of these proteins expressed in cells showed co-localization with endoplasmic reticulum (ER) markers. Further electron microscopy analysis of cells co-expressing FCV p39 or p30 with a horseradish peroxidase protein containing the KDEL ER retention motif demonstrated gross morphological changes to the ER. Similar reorganization patterns, especially for those produced by p30, were observed in naturally infected Crandel-Rees feline kidney cells. Together, the data demonstrate that the p32, p39 and p30 proteins of FCV locate to the ER and lead to reorganization of ER membranes. This suggests that they may play a role in the generation of FCV replication complexes and that the endoplasmic reticulum may represent the potential source of the membrane vesicles induced during FCV infection.

Influence of temperature on the composition and polymerization of gluten proteins during grain filling in spring wheat (Triticum aestivum L.)

One Norwegian and one UK spring wheat cultivar, Bjarne and Cadenza, respectively, were grown in climate chambers to investigate the effects of lower to moderate temperatures during grain filling on the gluten quality. Two experiments were carried out with weekly fertilization until anthesis, while post-anthesis fertilization was applied in a third experiment. The proportions of different gluten proteins were affected by temperature in a similar manner for both cultivars when grown without post-anthesis fertilization. However, whereas low temperature strongly decreased %UPP for Cadenza, Bjarne had high %UPP at all temperature regimes. The results indicated that the assembly of glutenin polymers in Bjarne was less sensitive to variation in temperature than in Cadenza. Thus, our results suggested that the temperature influenced the proportion of different gluten proteins in both cultivars, while its effects on the assembly of the glutenin polymers were cultivar dependent. The duration of grain filling was longer at the lower temperatures, and this was associated with increased grain weight. Temperature had little effect on the amount of protein accumulated per grain, thus the proportion of proteins was strongly decreased at lower temperatures. This was to some extent, but not fully counteracted by post-anthesis fertilization.

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.

Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.

Comparison of CLEC-2 and GPVI signaling in platelets: the role of adaptor proteins

GPVI activates platelets through an ITAM pathway by activation of Src and Syk kinases leading to activation of PLCy2. CLEC-2 has been shown to activate platelets using an ITAM-like sequence in its cytoplasmic tail that is also dependent on Src and Syk kinases, but shows a partial rather than an absolute dependence on adapter SLP-76 for activation of PLCy2. The aim of this thesis is to understand some of the key differences in these signalling pathways. GPVI is in complex with FcRwhich contains the ITAM sequence (Yxx(L/I)x6−12Yxx(L/I)). These two tyrosines provide a docking site for the tandem-SH2 domains of Syk. In this thesis I show that CLEC-2 signalling through Syk is mediated by phosphorylation of the CLEC-2 YxxL sequence, receptor dimerisation and cross-linking by the Syk SH2 domains. I also show that the differential requirement for SLP-76 is not mediated by Gads. Both signalling pathways also show partial dependency for LAT. I also show that a novel protein, G6f, is not able to substitute for LAT in this signalling pathway and also exclude the LAT-family proteins PAG, LIME, LAX and NTAL as potential LAT replacements in platelet activation by GPVI. These results extend our understanding of platelet activation by CLEC-2.

Regulation of actin assembly by SCAR/WAVE proteins.

Actin reorganization is a tightly regulated process that co-ordinates complex cellular events, such as cell migration, chemotaxis, phagocytosis and adhesion, but the molecular mechanisms that underlie these processes are not well understood. SCAR (suppressor of cAMP receptor)/WAVE [WASP (Wiskott-Aldrich syndrome protein)-family verprolin homology protein] proteins are members of the conserved WASP family of cytoskeletal regulators, which play a critical role in actin dynamics by triggering Arp2/3 (actin-related protein 2/3)-dependent actin nucleation. SCAR/WAVEs are thought to be regulated by a pentameric complex which also contains Abi (Abl-interactor), Nap (Nck-associated protein), PIR121 (p53-inducible mRNA 121) and HSPC300 (haematopoietic stem progenitor cell 300), but the structural organization of the complex and the contribution of its individual components to the regulation of SCAR/WAVE function remain unclear. Additional features of SCAR/WAVE regulation are highlighted by the discovery of other interactors and distinct complexes. It is likely that the combinatorial assembly of different components of SCAR/WAVE complexes will prove to be vital for their roles at the centre of dynamic actin reorganization.

Extractability and characteristics of proteins deriving from wheat DDGS

Wheat Distillers’ Dried Grains with Solubles (DDGS) and in-process samples were used for protein extraction. Prolamins were the predominant protein components in the samples. The absence of extractable α- and γ-gliadins in DDGS indicated protein aggregation during the drum drying processing stage. Prolamin extraction was performed using 70% (v/v) ethanol or alkaline-ethanol solution in the presence of reducing agent. DDGS extracts had relatively low protein contents (14-44.9%, w/w), regardless of the condition applied. The wet solids were the most suitable raw material for protein extraction, with recovery yields of ~ 55% (w/w) and protein content of ~58% (w/w) in 70% (v/v) ethanol. Protein extracts from wet solids were significantly rich in glutamic acid and proline. Mass balance calculations demonstrated the high carbohydrate content (~ 50%, w/w) of solid residues. Overall, the feasibility of utilising in-process samples of DDGS for protein extraction with commercial potential was demonstrated.

Proteins and their interacting partners: an introduction to protein–ligand binding site prediction methods

Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein–ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein–ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein–ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential.

Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress-induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H(2)O(2) induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H(2)O(2) stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from approximately 30 minutes). The levels of the mitochondrial membrane marker cytochrome oxidase remained unchanged. H(2)O(2) also induced translocation of cytochrome c from the mitochondria to the cytosol within 15 to 30 minutes, which was indicative of mitochondrial dysfunction. Myocytes exposed to H(2)O(2) showed an early loss of mitochondrial membrane potential (assessed by fluorescence-activated cell sorter analysis) from 15 to 30 minutes, which was partially restored by approximately 1 hour. However, a subsequent irreversible loss of mitochondrial membrane potential occurred that correlated with cell death. These data suggest that the regulation of Bcl-2 and mitochondrial function are important factors in oxidative stress-induced cardiac myocyte apoptosis.

Small guanine nucleotide-binding proteins and myocardial hypertrophy.

The small (21 kDa) guanine nucleotide-binding protein (small G protein) superfamily comprises 5 subfamilies (Ras, Rho, ADP ribosylation factors [ARFs], Rab, and Ran) that act as molecular switches to regulate numerous cellular responses. Cardiac myocyte hypertrophy is associated with cell growth and changes in the cytoskeleton and myofibrillar apparatus. In other cells, the Ras subfamily regulates cell growth whereas the Rho subfamily (RhoA, Rac1, and Cdc42) regulates cell morphology. Thus, the involvement of small G proteins in hypertrophy has become an area of significant interest. Hearts from transgenic mice expressing activated Ras develop features consistent with hypertrophy, whereas mice overexpressing RhoA develop lethal heart failure. In isolated neonatal rat cardiac myocytes, transfection or infection with activated Ras, RhoA, or Rac1 induces many of the features of hypertrophy. We discuss the mechanisms of activation of the small G proteins and the downstream signaling pathways involved. The latter may include protein kinases, particularly the mitogen-activated or Rho-activated protein kinases. We conclude that although there is significant evidence implicating Ras, RhoA, and Rac1 in hypertrophy, the mechanisms are not fully understood.

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Quantification of major camel milk proteins by capillary electrophoresis

Proteins from dromedary camel milk (CM) produced in Europe were separated and quantified by capillary electrophoresis (CE). CE analysis showed that camel milk lacks b-lactoglobulin and consists of high concentration of a-lactalbumin (2.01 ± 0.02 mg mL-1), lactoferrin (1.74 ± 0.06 mg mL-1) and serum albumin (0.46 ± 0.01 mg mL-1 ). Among caseins, the concentration of b-casein (12.78 ± 0.92 mg mL-1) was found the highest followed by a-casein (2.89 ± 0.29 mg mL-1) while k-casein represented only minor amount (1.67 ± 0.01 mg mL-1). These results were in agreement with sodium dodecyl sulphatepolyacrylamide gel electrophoresis patterns. Overall, CE offers a quick and reliable method for the determination of major CM proteins, which may be responsible for the many nutritional and health properties of CM.

Astringency reduction in red wine by whey proteins

Whey is a by-product of cheese manufacturing and therefore investigating new applications of whey proteins will contribute towards the valorisation of whey and hence waste reduction. This study shows for the first time a detailed comparison of the effectiveness of gelatin and β-lactoglobulin (β-LG) as fining agents. Gelatin was more reactive than whey proteins to tannic acid as shown by both the astringency method (with ovalbumin as a precipitant) and the tannins determination method (with methylcellulose as a precipitant). The two proteins showed similar selectivity for polyphenols but β-LG did not remove as much catechin. The fining agent was removed completely or to a trace level after centrifugation followed by filtration which minimises its potential allergenicity. In addition, improved understanding of protein–tannin interactions was obtained by fluorescence, size measurement and isothermal titration calorimetry (ITC). Overall this study demonstrates that whey proteins have the potential of reducing astringency in red wine and can find a place in enology.