Quantitative analysis of severe acute respiratory syndrome (SARS)-associated coronavirus-infected cells using proteomic approaches: implications for cellular responses to virus infection

We present the first proteomic analysis on the cellular response to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. The differential proteomes of Vero E6 cells with and without infection of the SARS-CoV were resolved and quantitated with two-dimensional differential gel electrophoresis followed by ESI-MS/MS identification. Moreover isotope-coded affinity tag technology coupled with two-dimensional LC-MS/MS were also applied to the differential proteins of infected cells. By combining these two complementary strategies, 355 unique proteins were identified and quantitated with 186 of them differentially expressed (at least 1.5-fold quantitative alteration) between infected and uninfected Vero E6 cells. The implication for cellular responses to virus infection was analyzed in depth according to the proteomic results. Thus, the present work provides large scale protein-related information to investigate the mechanism of SARS-CoV infection and pathogenesis.     

Proteomic analysis of hepatitis B virus-associated hepatocellular carcinoma: Identification of potential tumor markers

Hepatocellular carcinoma (HCC) is a malignancy of both underdeveloped and developing countries. Proteomes of ten pairs of clinical hepatitis B virus associated HCC tissue samples were obtained by high resolution two-dimensional gel electrophoresis. Comprehensive analyses of proteins associated with B-type HCC were focused on total differentially expressed proteins (> or = two-fold increase or decrease, Student's t-test, p < 0.05) from one pair of samples. Protein identification was done by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Comparative analyses of proteins associated with B-type HCC included repeat statistics in ten cases. A total of 100 protein spots, corresponding to 80 different gene products, were identified. Proteins whose expression levels were different by more than 2-fold in at least 50% of the cases (five of ten cases) were further analyzed and 45 proteins were selected out as candidates for HCC-associated proteins. Western blotting further validated up-regulated expressions of two candidate proteins in tumor tissues: proliferating cell antigen and stathmin 1. This comprehensive and comparative analyses of proteins associated with B-type HCC could provide useful molecular markers for diagnostics and prognostics and for therapeutic targets. The physiological significance of the differential expressions for several candidate proteins are discussed.     

Role of integrin-linked kinase in regulating phosphorylation of Akt and fibroblast survival in type I collagen matrices through a beta1 integrin viability signaling pathway

A beta1 integrin phosphatidylinositol 3-kinase/Akt pathway regulates fibroblast survival in collagen matrices. When fibroblasts attach to collagen, Akt becomes phosphorylated, providing a survival signal. In contrast, in response to mechanical forces generated during collagen contraction, Akt is dephosphorylated and fibroblasts undergo apoptosis. The kinase(s) responsible for regulating Akt phosphorylation in response to matrix-derived mechanical signals are unclear. Integrin-linked kinase (ILK) is associated with the beta1 integrin in the focal adhesion complex and as such is a candidate kinase that may regulate Akt phosphorylation and fibroblast viability. Nevertheless, there is no direct evidence that matrix-derived mechanical forces regulate cell viability by modulating ILK activity. Here, we show that ILK activity decreased in response to collagen matrix contraction, which correlated with Akt dephosphorylation and induction of fibroblast apoptosis. In contrast, enforced activation of beta1 integrin by activating antibody preserved ILK and Akt activity during collagen matrix contraction, and this is associated with protection from collagen contraction-induced apoptosis. Knock-down of ILK by small, interfering RNA (siRNA) attenuated Akt phosphorylation in response to ligation of beta1 integrin by collagen or activating antibody and enhanced fibroblast apoptosis in response to collagen contraction. Kinase dead ILK attenuated Akt phosphorylation and enhanced fibroblast apoptosis, whereas hyperactive and wild type ILK augmented Akt phosphorylation and protected fibroblasts from apoptosis. Constitutively active Akt preserved Akt activity and rescued ILK siRNA-treated fibroblasts from collagen contraction-induced apoptosis. These data establish that matrix-derived mechanical forces sensed by beta1 integrin are capable of modulating ILK activity which regulates fibroblast viability via an Akt-dependent mechanism.     

Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.     

Roles of 3-phosphoinositide-dependent kinase 1 in the regulation of endothelial nitric-oxide synthase phosphorylation and function by heat shock protein 90

The 90-kDa heat shock protein (Hsp90) plays an important role in endothelial nitric-oxide synthase (eNOS) regulation. Besides acting as an allosteric enhancer, Hsp90 was shown to serve as a module recruiting Akt to phosphorylate the serine 1179/1177 (bovine/human) residue of eNOS. Akt is activated by the phosphorylation of 3-phosphoinositide-dependent kinase 1 (PDK1). Whether PDK1 is involved in the actions of Hsp90 on eNOS phosphorylation and function remains unknown. To address this issue, we treated bovine eNOS stably transfected human embryonic kidney 293 cells with Hsp90 inhibitors and determined the alterations of phospho-eNOS, Akt, and PDK1. Both geldanamycin and radicicol, two structurally different Hsp90 inhibitors, selectively reduced serine 1179-phosphorylated eNOS, leading to decreased enzyme activity. In Hsp90-inhibited cells, eNOS-associated phospho-Akt was decreased, but the total amount of Akt associated with eNOS remained the same. Further studies showed that Hsp90 inhibition dramatically depleted intracellular PDK1. Proteasome but not caspase blockade prevented the loss of PDK1 caused by Hsp90 inhibition. Silencing the PDK1 gene by small interfering RNA was sufficient to induce reduction of phospho-Akt and consequent loss of serine 1179-phosphorylated eNOS. Moreover, overexpression of PDK1, but not Akt, reversed Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation and salvaged enzymatic activity. Thus, in addition to functioning as a module to recruit Akt to eNOS, Hsp90 also critically stabilized PDK1 by preventing it from proteasomal degradation. Inhibition of Hsp90 function resulted in PDK1 depletion and thus triggered a cascade of Akt deactivation, loss of eNOS serine 1179 phosphorylation, and decrease of enzyme function.     

Glutathionylation of two cysteine residues in paired domain regulates DNA binding activity of Pax-8

We reported that the first two cysteine residues out of three present in paired domain (PD), a DNA-binding domain, are responsible for redox regulation of Pax-8 DNA binding activity. We show that glutathionylation of these cysteines has a regulatory role in PD binding. Wild-type PD and its mutants with substitution of cysteine to serine were synthesized and named CCC, CSS, SCS, SSC, and SSS according to the positions of substituted cysteines. They were incubated in a buffer containing various ratios of GSH/GSSG and subjected to gel shift assay. Binding of CCC, CSS, and SCS was impaired with decreasing GSH/GSSG ratio, whereas that of SSC and SSS was not affected. Because [3H]glutathione was incorporated into CCC, CSS, and SCS, but not into SSC and SSS, the binding impairment was ascribed to glutathionylation of the redox-reactive cysteines. This oxidative inactivation of PD binding was reversed by a reductant dithiothreitol and by redox factor (Ref)-1 in vitro. To explore the glutathionylation in cells, Chinese hamster ovary cells overexpressing CSS and SCS were labeled with [35S]cysteine in the presence of cycloheximide. Immunoprecipitation with an antibody against PD revealed that treatment of the cells with an oxidant diamide induced the 35S incorporation into both mutants, suggesting the PD glutathionylation in cells. Since the two cysteine residues in PD are conserved in all Pax members, this novel posttranslational modification of PD would provide a new insight into molecular basis for modulation of Pax function.     

1H-NMR study of the effect of temperature through reversible unfolding on the heme pocket molecular structure and magnetic properties of aplysia limacina cyano-metmyoglobin

Two-dimensional 1H NMR spectroscopy over a range of temperature through thermal unfolding has been applied to the low-spin, ferric cyanide complex of myoglobin from Aplysia limacina to search for intermediates in the unfolding and to characterize the effect of temperature on the magnetic properties and electronic structure of the heme iron. The observation of strictly linear behavior from 5 to 80 C degrees through the unfolding transition for all hyperfine-shifted resonances indicates the absence of significant populations of intermediate states to the cooperative unfolding with Tm approximately 80 degrees C. The magnetic anisotropies and orientation of the magnetic axes for the complete range of temperatures were also determined for the complex. The anisotropies have very similar magnitudes, and exhibit the expected characteristic temperature dependence, previously observed in the isoelectronic sperm whale myoglobin complex. In contrast to sperm whale Mb, where the orientation of the magnetic axis was completely temperature-independent, the tilt of the major magnetic axis, which correlates with the Fe-CN tilt, decreases at high temperature in Aplysia limacina Mb, indicating a molecular structure that is conserved with temperature, although more plastic than that of sperm whale Mb. The pattern of contact shifts reflects a conserved Fe-His(F8) bond and pi-spin delocalization into the heme, as expected for the orientation of the axial His imidazole.     

Interaction between the movement protein of barley yellow dwarf virus and the cell nuclear envelope: role of a putative amphiphilic alpha-helix at the N-terminus of the movement protein

The open reading frame 4 (ORF 4) gene product of barley yellow dwarf virus (BYDV) may act as a movement protein (MP) by assisting the transport of viral genomic RNA across the nuclear envelope (NE) of host plant cells. To investigate interactions between BYDV MP and the NE, wild-type and mutant open reading frame (ORF 4)-green fluorescent protein (GFP) fusion cistrons were expressed in insect cells. A fusion protein expressed by the wild-type ORF 4-GFP cistron associated with the NE and caused protrusions from its surface. The fusion protein expressed by the mutant ORF 4-GFP cistron lacked a putative amphiphilic alpha-helix at its N-terminus and although associating with the NE, showed decreased levels of protrusions. A peptide homologue of this putative alpha-helix induced an increase of 7 degrees C in the phase transition temperature of dimyrystoyl phosphatidylserine (DMPS) membranes, accompanied by a decrease in membrane fluidity, but exhibited no significant interaction with either dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylethanolamine (DMPE) membranes. These results strongly support the view that BYDV MP may interact with the NE to help transport viral genomic RNA into the nuclear compartment. This function of BYDV MP appears to involve protrusions on the surface of the NE and may require the presence of an N-terminal amphiphilic alpha-helix, which is speculated to destabilize membranes, thereby assisting the entry of BYDV-GAV into the nuclear compartment.     

Disruption of Sertoli-germ cell adhesion function in the seminiferous epithelium of the rat testis can be limited to adherens junctions without affecting the blood-testis barrier integrity: an in vivo study using an androgen suppression model

During spermatogenesis, both adherens junctions (AJ) (such as ectoplasmic specialization (ES), a testis-specific AJ type at the Sertoli cell-spermatid interface (apical ES) or Sertoli-Sertoli cell interface (basal ES) in the apical compartment and BTB, respectively) and tight junctions (TJ) undergo extensive restructuring to permit germ cells to move across the blood-testis barrier (BTB) as well as the seminiferous epithelium from the basal compartment to the luminal edge to permit fully developed spermatids (spermatozoa) to be sloughed at spermiation. However, the integrity of the BTB cannot be compromised throughout spermatogenesis so that postmeiotic germ cell-specific antigens can be sequestered from the systemic circulation at all times. We thus hypothesize that AJ disruption in the seminiferous epithelium unlike other epithelia, can occur without compromising the BTB-barrier, even though these junctions, namely TJ and basal ES, co-exist side-by-side in the BTB. Using an intratesticular androgen suppression-induced germ cell loss model, we have shown that the disruption of AJs indeed was limited to the Sertoli-germ cell interface without perturbing the BTB. The testis apparently is using a unique physiological mechanism to induce the production of both TJ- and AJ-integral membrane proteins and their associated adaptors to maintain BTB integrity yet permitting a transient loss of cell adhesion function by dissociating N-cadherin from beta-catenin at the apical and basal ES. The enhanced production of TJ proteins, such as occludin and ZO-1, at the BTB site can supersede the transient loss of cadherin-catenin function at the basal ES. This thus allows germ cell depletion from the epithelium without compromising BTB integrity. It is plausible that the testis is using this novel mechanism to facilitate the movement of preleptotene and leptotene spermatocytes across the BTB at late stage VIII through early stage IX of the epithelial cycle in the rat while maintaining the BTB immunological barrier function.