Extracellular disulfide exchange and the regulation of cellular function

An emerging concept is that disulfide bonds can act as a dynamic scaffold to present mature proteins in different conformational and functional states on the cell surface. Two examples are the conversion of the receptor, integrin a alpha(IIb)beta(3), from a low affinity to a high affinity state, and the interaction of CD4 receptor with the HIV-1 envelope glycoprotein gp120 to promote virus-cell fusion. In both of these cases there is a remodeling of the protein disulfide bonding pattern. The formation and rearrangement of disulfide bonds is modulated by a family of enzymes known as the thiol isomerases, which include protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72. While these enzymes were reported originally to be restricted in location to the endoplasmic reticulum, in some cells thiol isomerases are found on the cell surface. This may indicate a wider role for these enzymes in cell function. In platelets it has been shown that reagents that react with cell surface sulfhydryl groups are capable of blocking a number of functional responses, including integrin-mediated aggregation, adhesion, and granule secretion. Furthermore, the use of function blocking antibodies to either PDI or ERp5 causes inhibition of these functional responses. This review summarizes current knowledge of the extracellular regulation of disulfide exchange and the implications of this in the regulation of cell function.

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Regulation of SHP-1 tyrosine phosphatase in human platelets by serine phosphorylation at its C terminus

SHP-1 is a Src homology 2 (SH2) domain-containing tyrosine phosphatase that plays an essential role in negative regulation of immune cell activity. We describe here a new model for regulation of SHP-1 involving phosphorylation of its C-terminal Ser(591) by associated protein kinase Calpha. In human platelets, SHP-1 was found to constitutively associate with its substrate Vav1 and, through its SH2 domains, with protein kinase Calpha. Upon activation of either PAR1 or PAR4 thrombin receptors, the association between the three proteins was retained, and Vav1 became phosphorylated on tyrosine and SHP-1 became phosphorylated on Ser(591). Phosphorylation of SHP-1 was mediated by protein kinase C and negatively regulated the activity of SHP-1 as demonstrated by a decrease in the in vitro ability of SHP-1 to dephosphorylate Vav1 on tyrosine. Protein kinase Calpha therefore critically and negatively regulates SHP-1 function, forming part of a mechanism to retain SHP-1 in a basal active state through interaction with its SH2 domains, and phosphorylating its C-terminal Ser(591) upon cellular activation leading to inhibition of SHP-1 activity and an increase in the tyrosine phosphorylation status of its substrates.

Identification of a serine protease involved with the regulation of adrenal growth

The adrenal cortex is a dynamic organ in which the cells of the outer cortex continually divide. It is well known that this cellular proliferation is dependent on constant stimulation from peptides derived from the ACTH precursor pro-opiomelanocortin (POMC) because disruption of pituitary corticotroph function results in rapid atrophy of the gland. Previous results from our laboratory have suggested that the adrenal mitogen is a fragment derived from the N-terminal of POMC not containing the gamma-MSH sequence. Because such a peptide is not generated during processing of POMC in the pituitary, we proposed that the mitogen is generated from circulating pro-gamma-MSH by an adrenal protease. Using degenerate oligonucleotides, we identified a secreted serine protease expressed by the adrenal gland that we named adrenal secretory protease (ASP). In the adrenal cortex, expression of ASP is limited to the outer zona glomerulosa/fasciculata, the region where cortical cells are believed to be derived, and is significantly up-regulated during compensatory growth. Y1 adrenocortical cells transfected with a vector expressing an antisense RNA (and thus having reduced levels of endogenous ASP) were found to grow slower than sense controls while also losing their ability to utilize exogenous pro-gamma-MSH in the media supporting a role for ASP in adrenal growth. Digestion of an N-POMC peptide substrate encompassing the residues around the dibasic cleavage site at positions 49/50 with affinity-purified ASP showed cleavage not to occur at the dibasic site but two residues downstream leading us to propose the identity of the adrenal mitogen to be N-POMC (1-52).

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Fatty acids as gatekeepers of immune cell regulation

Fatty acids have diverse roles in all cells. They are important as a source of energy, as structural components of cell membranes, as signalling molecules and as precursors for the synthesis of eicosanoids. Recent research has suggested that the organization of fatty acids into distinct cellular pools has a particularly important role in cells of the immune system and that forms of lipid trafficking exist, which are as yet poorly understood. This Review examines the nature and regulation of cellular lipid pools in the immune system, their delivery of fatty acids or fatty acid derivatives to specific locations and their potential role in health and disease.

Inhibition of cellular proliferation by the genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone is mediated by DNA damage and activation of the ATR signalling pathway

The cellular actions of genistein, and its in vivo metabolites, are believed to mediate the decreased risk of breast cancer associated with high soy consumption. The genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone (THIF), induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells via a mechanism involving the activation of ataxia telangiectasia and Rad3-related kinase (ATR) via its phosphorylation at Ser(428). This activation of ATR appeared to result from THIF-induced increases in intracellular oxidative stress, a depletion of cellular GSH and an increase in DNA strand breakage. THIF treatment also led to an inhibition of cdc2, which was accompanied by the phosphorylation of both p53 (Ser(15)) and Chk1 (Ser(296)) and the de-activation of cdc25C phosphatase. We suggest the anti-proliferative actions of THIF may be mediated by initial oxidative DNA damage, activation of ATR and downstream regulation of the p53 and Chk1 pathways leading to cell cycle arrest in G2-M. This may represent one mechanism by which genistein exerts its cellular activity in vivo. (c) 2007 Elsevier Inc. All rights reserved.

Regulation of cell cycle and stress responses to hydrostatic pressure in fission yeast

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress.

Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin

Background: Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). Results: Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region. Conclusions: Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

Translationally controlled tumour protein (TCTP) is a highly conserved protein present in all eukaryotic organisms. Various cellular functions and molecular interactions have been ascribed to this protein, many related to its growth-promoting and antiapoptotic properties. TCTP levels are highly regulated in response to various cellular stimuli and stresses. We have shown recently that the double-stranded RNA-dependent protein kinase, PKR, is involved in translational regulation of TCTP. Here we extend these studies by demonstrating that TCTP is downregulated in response to various proapoptotic treatments, in particular agents that induce Ca++ stress, in a PKR-dependent manner. This regulation requires phosphorylation of protein synthesis factor eIF2α. Since TCTP has been characterized as an antiapoptotic and Ca++-binding protein, we asked whether it is involved in protecting cells from Ca++-stress-induced apoptosis. Overexpression of TCTP partially protects cells against thapsigargin-induced apoptosis, as measured using caspase-3 activation assays, a nuclear fragmentation assay, using fluorescence-activated cell sorting analysis, and time-lapse video microscopy. TCTP also protects cells against the proapoptotic effects of tunicamycin and etoposide, but not against those of arsenite. Our results imply that cellular TCTP levels influence sensitivity to apoptosis and that PKR may exert its proapoptotic effects at least in part through downregulation of TCTP via eIF2α phosphorylation.

Nuclear Dbf2-related protein kinases (NDRs) in isolated cardiac myocytes and the myocardium: activation by cellular stresses and by phosphoprotein serine-/threonine-phosphatase inhibitors

The nuclear Dbf2-related protein kinases 1 and 2 (NDR1/2) are closely-related AGC family kinases that are strongly conserved through evolution. In mammals, they are activated inter alia by phosphorylation of an hydrophobic domain threonine-residue [NDR1(Thr-444)/NDR2(Thr-442)] by an extrinsic protein kinase followed by autophosphorylation of a catalytic domain serine-residue [NDR1(Ser-281)/NDR2(Ser-282)]. We examined NDR1/2 expression and regulation in primary cultures of neonatal rat cardiac myocytes and in perfused adult rat hearts. In myocytes, transcripts for NDR2, but not NDR1, were induced by the hypertrophic agonist, endothelin-1. NDR1(Thr-444) and NDR2(Thr-442) were rapidly phosphorylated (maximal in 15-30 min) in myocytes exposed to some phosphoprotein Ser-/Thr-phosphatase 1/2 inhibitors (calyculin A, okadaic acid) and, to a lesser extent, by hyperosmotic shock, low concentrations of H(2)O(2), or chelerythrine. In myocytes adenovirally-transduced to express FLAG-NDR2 (which exhibited a mainly-cytoplasmic localisation), the same agents increased FLAG-NDR2 activity as assessed by in vitro protein kinase assays, indicative of FLAG-NDR2(Ser-282/Thr-442) phosphorylation. Calyculin A-induced phosphorylation of NDR1(Thr-444)/NDR2(Thr-442) and activation of FLAG-NDR2 were inhibited by staurosporine, but not by other protein kinase inhibitors tested. In ex vivo rat hearts, NDR1(Thr-444)/NDR2(Thr-442) were phosphorylated in response to ischaemia-reperfusion or calyculin A. From a pathological viewpoint, we conclude that activities of NDR1 and NDR2 are responsive to cytotoxic stresses in heart preparations and this may represent a previously-unidentified response to myocardial ischaemia in vivo.

Glycogen synthase kinases 3α and 3β in cardiac myocytes: regulation and consequences of their inhibition

Inhibition of glycogen synthase kinase 3β (GSK3β) as a consequence of its phosphorylation by protein kinase B/Akt (PKB/Akt) has been implicated in cardiac myocyte hypertrophy in response to endothelin-1 or phenylephrine. We examined the regulation of GSK3α (which we show to constitute a significant proportion of the myocyte GSK3 pool) and GSK3β in cardiac myocytes. Although endothelin increases phosphorylation of GSK3 and decreases its activity, the response is less than that induced by insulin (which does not promote cardiac myocyte hypertrophy). GSK3 phosphorylation induced by endothelin requires signalling through the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and not the PKB/Akt pathway, whereas the reverse is true for insulin. Cardiac myocyte hypertrophy involves changes in morphology, and in gene and protein expression. The potent GSK3 inhibitor 1-azakenpaullone increases myocyte area as a consequence of increased cell length whereas phenylephrine increases both length and width. Azakenpaullone or insulin promotes AP1 transcription factor binding to an AP1 consensus oligonucleotide, but this was significantly less than that induced by endothelin and derived principally from increased binding of JunB protein, the expression of which was increased. Azakenpaullone promotes significant changes in gene expression (assessed by Affymetrix microarrays), but the overall response is less than with endothelin and there is little overlap between the genes identified. Thus, although GSK3 may contribute to cardiac myocyte hypertrophy in some respects (and presumably plays an important role in myocyte metabolism), it does not appear to contribute as significantly to the response induced by endothelin as has been maintained.

Cannabis sativa and the endogenous cannabinoid system: therapeutic potential for appetite regulation

The herb Cannabis sativa (C. sativa) has been used in China and on the Indian subcontinent for thousands of years as a medicine. However, since it was brought to the UK and then the rest of the western world in the late 19th century, its use has been a source of controversy. Indeed, its psychotropic side effects are well reported but only relatively recently has scientific endeavour begun to find valuable uses for either the whole plant or its individual components. Here, we discuss evidence describing the endocannabinoid system, its endogenous and exogenous ligands and their varied effects on feeding cycles and meal patterns. Furthermore we also critically consider the mounting evidence which suggests non‐tetrahydrocannabinol phytocannabinoids play a vital role in C. sativa‐induced feeding pattern changes. Indeed, given the wide range of phytocannabinoids present in C. sativa and their equally wide range of intra‐, inter‐ and extra‐cellular mechanisms of action, we demonstrate that non‐Δ9tetrahydrocannabinol phytocannabinoids retain an important and, as yet, untapped clinical potential.