A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells

The plant hormone abscisic acid (ABA) serves as a physiological monitor to assess the water status of plants and, under drought conditions, induces stomatal pore closure by activating specific ion channels, such as a slow-anion channel (SLAC1) that, in turn, mediate ion efflux from the guard cells. Earlier genetic analyses uncovered a protein kinase (OST1) and several 2C-type phosphatases, as respective positive and negative regulators of ABA-induced stomatal closure. Here we show that the OST1 kinase interacts with the SLAC1 anion channel, leading to its activation via phosphorylation. PP2CA, one of the PP2C phosphatase family members acts in an opposing manner and inhibits the activity of SLAC1 by two mechanisms: (1) direct interaction with SLAC1 itself, and (2) physical interaction with OSTI leading to inhibition of the kinase independently of phosphatase activity. The results suggest that ABA signaling is mediated by a physical interaction chain consisting of several components, including a PP2C member, SnRK2-type kinase (OST1), and an ion channel, SLAC1, to regulate stomatal movements. The findings are in keeping with a paradigm in which a protein kinase-phosphatase pair interacts physically with a target protein to couple a signal with a specific response.     

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor

Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell–cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell–cell contact by global analysis of protein phosphorylation. To define immediate CD28 targets, we used phosphorylated forms of the CD28 cytoplasmic region to obtain the CD28 interactome. The interaction profiles of selected CD28-interacting proteins were further characterized in vivo for amplifying the CD28 interactome. The combination of the global phosphorylation and interactome analyses revealed broad regulation of CD28 and its interactome by phosphorylation. Among the cellular phosphoproteins influenced by CD28 signaling, CapZ-interacting protein (CapZIP), a regulator of the actin cytoskeleton, was implicated by functional studies. The combinatorial approach applied herein is widely applicable for characterizing signaling networks associated with membrane receptors with short cytoplasmic tails.During some forms of intercellular communication, soluble growth factors are secreted by one cell type and bind specific transmembrane receptor tyrosine kinases (RTKs) present on neighboring cells. Activated RTKs then undergo autophosphorylation and also phosphorylate associated scaffold proteins, thereby creating docking sites for effector proteins with phosphotyrosine (pTyr)-recognition modules such as Src homology 2 (SH2) and pTyr-binding (PTB) domains (1). These immediate targets of growth-factor receptors control a variety of intracellular responses, typified by the activation of serine/threonine protein kinases such as those involved in the ERK MAP kinase pathway. Growth-factor signaling therefore leads to extensive changes in both tyrosine and serine/threonine phosphorylation (2).The alterations in protein–protein interactions and posttranslational modifications elicited by transmembrane receptors can in principle be characterized by mass spectrometry (MS)-based proteomics (3–6). However, under circumstances that involve complex cell–cell interactions, identifying physiologically relevant signals using MS-based proteomics can be challenging. For example, obtaining a sufficiently robust response for MS analysis has often required the use of cells that overexpress a receptor of interest and are subjected to a nonphysiological stimulus. For this reason, experiments have often focused on specific, often stereotypic, aspects of the cellular response, rather than taking a more global approach. Moreover, cell–cell interactions are often typified by the interaction of multiple transmembrane receptor-ligand pairs, frequently involving membrane proteins that are not receptor kinases. Short-peptide motifs from such receptors that serve as potential ligands for downstream targets can be used as affinity probes for binding partners, but these experiments suffer from high false-positive and false-negative rates (7, 8). Importantly, existing techniques are especially unsuitable for investigating the common situation typified by cells of the immune system, in which antigen-dependent signaling is induced by direct cell–cell contact, rather than by soluble secreted factors.This latter issue is exemplified by the observation that communication between two distinct cell types often can be controlled by the interaction of two transmembrane or membrane-tethered proteins (9). Under these circumstances, investigating signaling events requires that cells expressing an activating membrane-associated ligand are cocultured with cells displaying its corresponding receptor. However, exploring the resulting signal transduction pathways is challenging because the identity of the cell from which a particular protein is derived is lost after cell lysis. The T.P. laboratory developed a proteomic approach [termed quantitative analysis of bidirectional signaling (qBidS)] for profiling cell-specific tyrosine phosphorylation events resulting from contact between cells expressing either an Eph receptor tyrosine kinase or its ephrin ligand, respectively (10). In these experiments, the EphB2 receptor or its ephrin-B1 ligand, a transmembrane protein that itself has intrinsic signaling activity, was stably expressed in human embryonic kidney (HEK) 293 cells so that the two cell types segregated in culture due to repulsive EphB2/ephrin-B1 signaling. The two distinct cell populations were then differentially labeled with stable isotopes [stable isotope labeling by amino acid in cell culture (SILAC)] and cocultured. This strategy not only can identify tyrosine phosphorylation events modulated after the interaction between EphB2 and ephrin-B1 but also can indicate from which cell type a specific phosphopeptide originates, and thus can infer pathways activated in either cell type involved in EphB2/ephrin-B1 bidirectional signaling. However, these experiments have limitations. One is that they depend on overexpression of receptors and ligands in a model-cell system. Another is that, once two cells adhere to one another, they may interact through multiple different ligands and receptors, making it difficult to confidently define the exclusive signaling output of a single class of receptor (9, 11).Here, we overcome these issues by using a combination of proteomic and cell-biological approaches to systematically identify specific signaling events downstream of the endogenous CD28 costimulatory receptor on T cells. CD28 is a type I transmembrane protein that binds through its extracellular region to B7 proteins (CD80 and CD86), which are transmembrane proteins expressed on the surface of antigen-presenting cells (APCs) and are up-regulated by inflammatory signals. Upon the interaction of T cells and APCs, the T-cell receptor (TCR) engages a peptide antigen-bound major histocompatibility complex molecule on the APCs, resulting in a primary signal. Simultaneously, costimulatory signaling is established predominantly, but not exclusively, through the association of CD28 on T cells with B7 ligands on the surface of the APCs. Both TCR and CD28 signals are required for a full T-cell response, resulting in, for example, production of the IL-2 cytokine. Although it lacks an intrinsic catalytic domain, the short 41 amino acid cytoplasmic tail of CD28 has highly conserved tyrosine-based motifs that are phosphorylated by cytoplasmic tyrosine kinases upon T-cell stimulation and that bind targets with SH2 domains in a pTyr-dependent manner, as well as proline-rich sequences that potentially bind SH3 domains (12). It therefore can help orchestrate the response to the signal from an APC, such as a B cell.Because the complex cellular interactions of T cells and APCs stimulate multiple receptors (e.g., the integrin receptor) in addition to CD28, we have developed techniques to distinguish specific events uniquely dependent solely on CD28 signaling to undertake a comprehensive screen of tyrosine and serine/threonine phosphorylation sites selectively regulated by CD28. We have used a complementary approach using the full cytoplasmic tail of CD28 to identify the cytoplasmic targets with which it interacts. This approach involved immunoprecipitation coupled to mass spectrometry (IP-MS) to identify a network of interacting proteins immediately proximal to CD28. We then integrated these data to dissect CD28-based signaling networks activated in T cells after coculture with APCs. We targeted one newly identified CD28-regulated phosphoprotein, CapZ-interacting protein (CapZIP), to demonstrate its importance in the CD28-dependent functional response. Our results demonstrate the value of a combinatorial and broad-based strategy to identify signaling pathways regulated by a specific endogenous receptor activated by direct cell–cell contact.     

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements.     

Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis

Phosphorylation of the cytoplasmic tyrosine residues of the epidermal growth factor receptor (EGFR) upon binding of EGF induces recognition of various intracellular signaling molecules, including Grb2. Here, the reaction kinetics between EGFR and Grb2 was analyzed by visualizing single molecules of Grb2 conjugated to the fluorophore Cy3 (Cy3-Grb2). The plasma membrane fraction was purified from human epithelial carcinoma A431 cells after stimulation with EGF and attached to coverslips. Unitary events of association and dissociation of Cy3-Grb2 on the EGFR in the membrane fraction were observed at different concentrations of Grb2 (0.1-100 nM). The dissociation kinetics could be explained by using a multiple-exponential function with a major (>90%) dissociation rate of 8 s(-1) and a few minor components, suggesting the presence of multiple bound states. In contrast, the association kinetics could be described by a stretched exponential function, suggesting the presence of multiple reaction channels from many unbound substates. Transitions between the unbound substates were also suggested. Unexpectedly, the rate of association was not proportional to the Grb2 concentration: an increase in Cy3-Grb2 concentration by a factor of 10 induced an increase in the reaction frequency approximately by a factor of three. This effect can compensate for fluctuation of the signal transduction from EGFR to Grb2 caused by variations in the expression level of Grb2 in living cells.     

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Newly synthesized cellular proteins can be tagged with a variety of metabolic labels that distinguish them from preexisting proteins and allow them to be identified and tracked. Many such labels are incorporated into proteins via the endogenous cellular machinery and can be used in numerous cell types and organisms. Though broad applicability has advantages, we aimed to develop a strategy to restrict protein labeling to specified mammalian cells that express a transgene. Here we report that heterologous expression of a mutant methionyl-tRNA synthetase from Escherichia coli permits incorporation of azidonorleucine (Anl) into proteins made in mammalian (HEK293) cells. Anl is incorporated site-selectively at N-terminal positions (in competition with initiator methionines) and is not found at internal sites. Site selectivity is enabled by the fact that the bacterial synthetase aminoacylates mammalian initiator tRNA, but not elongator tRNA. N-terminally labeled proteins can be selectively conjugated to a variety of useful probes; here we demonstrate use of this system in enrichment and visualization of proteins made during various stages of the cell cycle. N-terminal incorporation of Anl may also be used to engineer modified proteins for therapeutic and other applications.     

PI3K/AKT pathway activation in acute myeloid leukaemias is not associated with AKT1 pleckstrin homology domain mutation

Despite its' central role, the precise mechanisms of the phosphoinositide 3-kinase/Akt (PI3K)/Akt pathway activation in acute myeloid leukaemia (AML) have not been elucidated. Recently, a recurrent novel AKT1 pleckstrin homology domain (PHD) mutation leading to membrane translocation, constitutive AKT activation and leukaemia development in mice was described. To assess AKT1 PHD mutations in AML, we sequenced 57 specimens from 49 AML patients, all of whom showed PI3K/AKT pathway activation by analysis of total and phospho-protein expression for AKT, mTor, p70S6Kinase, S6ribosomal protein and PTEN. No mutations in AKT1 PHD were identified, making this mutation an unlikely cause of PI3K/AKT pathway activation in AML.     

A proteomic approach to identify proteins from Trichuris trichiura extract with immunomodulatory effects

Infections with Trichuris trichiura and other trichurid nematodes have been reported to display protective effects against atopy, allergic and autoimmune diseases. The aims of the present study were to investigate the immunomodulatory properties of T. trichiura adult worm extract (TtE) and its fractions (TtEFs) on the production of cytokines by peripheral blood mononuclear cells and to identify their proteinaceous components. Fourteen TtEFs were obtained by ion exchange chromatography and tested for effects on cytokine production by peripheral blood mononuclear cells. The molecular constituents of the six most active fractions were evaluated using nano‐LC/mass spectrometry. The homology between T. trichiura and the related nematode Trichinella spiralis was used to identify 12 proteins in TtEFs. Among those identified, fructose biphosphate aldolase, a homologue of macrophage migration inhibitory factor and heat‐shock protein 70 may contribute to the immunomodulatory effects of TtEFs. The identification of such proteins could lead to the development of novel drugs for the therapy of allergic and other inflammatory diseases.     

Leptin promotes the tyrosine phosphorylation of SHC proteins and SHC association with GRB2

The identification and characterization of proteins that become tyrosine phosphorylated in response to growth factor stimulation is critical for furthering our understanding of the signal transduction pathways involved in the regulation of cell proliferation, differentiation as well as metabolic activities. In this report, we demonstrate for the first time, that leptin is able to induce the tyrosine phosphorylation of the SH₂ containing protein SHC. These studies have been carried out on a human embryonic cell line (HEK 293) transfected with the cDNA encoding for the long form of the leptin receptor and stably expressing the receptor itself. We also shown that upon tyrosine phosphorylation, SHC associated with the adaptor protein, Grb₂. The formation of this complex may directly link tyrosine phosphorylation events to Ras activation and may be a critical step in proliferation and/or differentiation of cells. In conclusion, these results indicate that leptin receptor, after binding the ligand, activates several pathways for signal transduction that might lead to mitogenic effect.     

Detection of a ternary complex of NF-kappaB and IkappaBalpha with DNA provides insights into how IkappaBalpha removes NF-kappaB from transcription sites

It has been axiomatic in the field of NF-κB signaling that the formation of a stable complex between NF-κB and the ankyrin repeat protein IκBα precludes the interaction of NF-κB with DNA. Contradicting this assumption, we present stopped-flow fluorescence and NMR experiments that give unequivocal evidence for the presence of a ternary DNA-NF-κB-IκBα complex in solution. Stepwise addition of a DNA fragment containing the κB binding sequence to the IκBα-NF-κB complex results in changes in the IκBα NMR spectrum that are consistent with dissociation of the region rich in proline, glutamate, serine, and threonine (PEST) and C-terminal ankyrin repeat sequences of IκBα from the complex. However, even at high concentrations of DNA, IκBα remains associated with NF-κB, indicated by the absence of resonances of the free N-terminal ankyrin repeats of IκBα. The IκBα-mediated release of NF-κB from its DNA-bound state may be envisioned as the reverse of this process. The initial step would consist of the coupled folding and binding of the intrinsically disordered nuclear localization sequence of the p65 subunit of NF-κB to the well-structured N-terminal ankyrin repeats of IκBα. Subsequently the poorly folded C-terminal ankyrin repeats of IκBα would fold upon binding to the p50 and p65 dimerization domains of NF-κB, permitting the negatively charged C-terminal PEST sequence of IκBα to displace the bound DNA through a process of local mass action.     

Tyrosine phosphorylation sites on FRS2alpha responsible for Shp2 recruitment are critical for induction of lens and retina

Early development of the lens and retina depends upon reciprocal inductive interactions between the embryonic surface ectoderm and the underlying neuroepithelium of the optic vesicle. FGF signaling has been implicated in this signal exchange. The docking protein FRS2α is a major mediator of FGF signaling by providing a link between FGF receptors (FGFRs) and a variety of intracellular signaling pathways. After FGF stimulation, tyrosine-phosphorylated FRS2α recruits four molecules of the adaptor protein Grb2 and two molecules of the protein tyrosine phosphatase Shp2, resulting in activation of the Ras/extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase/Akt signaling pathways. In this report, we explore the role of signaling pathways downstream of FRS2α in eye development by analyzing the phenotypes of mice that carry point mutations in either the Grb2-(Frs2α^4F) or the Shp2-binding sites (Frs2α^2F) of FRS2α. Although Frs2α^4F/4F mice exhibited normal early eye development, all Frs2α^2F/2F embryos were defective in eye development and showed anophthalmia or microphthalmia. Consistent with the critical role of FRS2α in FGF signaling, the level of activated extracellular signal-regulated kinase in Frs2α^2F/2F embryos was significantly lower than that observed in wild-type embryos. Furthermore, expression of Pax6 and Six3, molecular markers for lens induction, were decreased in the Frs2α^2F/2F presumptive lens ectoderm. Similarly, the expression of Chx10 and Bmp4, genes required for retinal precursor proliferation and for lens development, respectively, was also decreased in the optic vesicles of Frs2α^2F/2F mice. These experiments demonstrate that intracellular signals that depend on specific tyrosine residues in FRS2α lie upstream of gene products critical for induction of lens and retina.     

Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

Ectokinases can phosphorylate extracellular proteins and external domains of membrane proteins influencing cell adhesion, movement, and cellular interactions. An ectokinase with the properties of casein kinase 2 (CK2) has been previously described, but little is known about the structural characteristics that allow this enzyme to be exported from the cell. Transfection of human embryonic kidney-293 cells with cDNAs coding for the catalytic (CK2alpha or CK2alpha') and regulatory (CK2beta) subunits with hemaglutinin tags allowed us to study the export of ectopically synthesized enzyme. When the catalytic (CK2alpha or CK2alpha') and the CK2beta regulatory subunits are cotransfected, the tetrameric enzyme composed of both subunits (holoenzyme) is detected outside the cell. This observation has been confirmed by assaying protein kinase activity in immunoprecipitates obtained with antihemaglutinin antibody by using a CK2-specific peptide substrate and by Western blots as well as by immunofluorescence of nonpermeabilized cells. Transfection with cDNA of catalytic or regulatory subunit alone does not result in export of these subunits. A study of the kinetics of appearance of the ectopically synthesized protein at different times after transfection indicates that a 5- to 7-h delay after the synthesis of the protein before it appears in the extracellular compartment. Using mutations of CK2alpha that eliminate phosphorylating activity [CK2alpha(Asp-156-Ala)] or that make it less sensitive to heparin inhibition [CK2alpha(Lys-75-Glu,Lys-76-Glu)] demonstrated that these mutations do not prevent the holoenzyme to be exported from the cells.     

APP17肽对DM大鼠海马神经元胰岛素和凋亡信号传导通路相关蛋白的影响

目的 观察糖尿病大鼠海马脑区胰岛素和凋亡信号传导通路中某些相关蛋白的表达及APP17肽对其表达的影响。方法 链脲佐菌素诱发大鼠糖尿病模型，并用APP17肽治疗。用免疫沉淀和Western-blot法分析海马中信号传导及部分凋亡相关蛋白；电镜观察大鼠脑组织超微结构。结果 DM大鼠IGF－IRα、Akt/PKB、CREB表明明显降低，APP17肽治疗后明显上调（P＜0．05），GSK－3β、PP－1及凋亡相关蛋白Bax、AIF表达增加，治疗后则明显降低（P＜0．05）；超微结构表明APP17肽治疗后兴奋性突触小泡明显减少。结论 糖尿病大鼠海马脑区胰岛素和凋亡信号传导通路中某些重要蛋白表达异常，APP17肽部分纠正这些蛋白的异常表达，这可能是其保护DM大鼠海马神经元的机制。     

Expression, signaling proficiency, and stimulatory function of the NKG2D lymphocyte receptor in human cancer cells

The stimulatory natural killer group 2 member D (NKG2D) lymphocyte receptor and its tumor-associated ligands are important mediators in the immune surveillance of cancer. With advanced human tumors, however, persistent NKG2D ligand expression may favor tumor progression. We have found that cancer cells themselves express NKG2D in complex with the DNAX-activating protein 10 (DAP10) signaling adaptor. Triggering of NKG2D on ex vivo cancer cells or on tumor lines which express only few receptor complexes activates the oncogenic PI3K-protein kinase B (PKB/AKT)-mammalian target of rapamycin (mTOR) signaling axis and downstream effectors, the ribosomal protein S6 kinase 1 (S6K1) and the translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, as in lymphocytes, NKG2D ligand engagement stimulates phosphorylation of JNK and ERK in MAP kinase cascades. Consistent with these signaling activities, above-threshold expression of NKG2D-DAP10 in a ligand-bearing tumor line increases its bioenergetic metabolism and proliferation, thus suggesting functional similarity between this immunoreceptor and tumor growth factor receptors. This relationship is supported by significant correlations between percentages of cancer cells that are positive for surface NKG2D and criteria of tumor progression. Hence, in a conceptual twist, these results suggest that tumor co-option of NKG2D immunoreceptor expression may complement the presence of its ligands for stimulation of tumor growth.     

Augmented glucose-induced insulin release in mice lacking G(o2), but not G(o1) or G(i) proteins

Insulin secretion by pancreatic β cells is a complex and highly regulated process. Disruption of this process can lead to diabetes mellitus. One of the various pathways involved in the regulation of insulin secretion is the activation of heterotrimeric G proteins. Bordetella pertussis toxin (PTX) promotes insulin secretion, suggesting the involvement of one or more of three G(i) and/or two G(o) proteins as suppressors of insulin secretion from β cells. However, neither the mechanism of this inhibitory modulation of insulin secretion nor the identity of the G(i/o) proteins involved has been elucidated. Here we show that one of the two splice variants of G(o), G(o2), is a key player in the control of glucose-induced insulin secretion by β cells. Mice lacking G(o2)α, but not those lacking α subunits of either G(o1) or any G(i) proteins, handle glucose loads more efficiently than wild-type (WT) mice, and do so by increased glucose-induced insulin secretion. We thus provide unique genetic evidence that the G(o2) protein is a transducer in an inhibitory pathway that prevents damaging oversecretion of insulin.     

Parallel adaptive feedback enhances reliability of the Ca2+ signaling system

Despite large cell-to-cell variations in the concentrations of individual signaling proteins, cells transmit signals correctly. This phenomenon raises the question of what signaling systems do to prevent a predicted high failure rate. Here we combine quantitative modeling, RNA interference, and targeted selective reaction monitoring (SRM) mass spectrometry, and we show for the ubiquitous and fundamental calcium signaling system that cells monitor cytosolic and endoplasmic reticulum (ER) Ca(2+) levels and adjust in parallel the concentrations of the store-operated Ca(2+) influx mediator stromal interaction molecule (STIM), the plasma membrane Ca(2+) pump plasma membrane Ca-ATPase (PMCA), and the ER Ca(2+) pump sarco/ER Ca(2+)-ATPase (SERCA). Model calculations show that this combined parallel regulation in protein expression levels effectively stabilizes basal cytosolic and ER Ca(2+) levels and preserves receptor signaling. Our results demonstrate that, rather than directly controlling the relative level of signaling proteins in a forward regulation strategy, cells prevent transmission failure by sensing the state of the signaling pathway and using multiple parallel adaptive feedbacks.     

Systematic identification of interactions between host cell proteins and E7 oncoproteins from diverse human papillomaviruses

More than 120 human papillomaviruses (HPVs) have now been identified and have been associated with a variety of clinical lesions. To understand the molecular differences among these viruses that result in lesions with distinct pathologies, we have begun a MS-based proteomic analysis of HPV-host cellular protein interactions and have created the plasmid and cell line libraries required for these studies. To validate our system, we have characterized the host cellular proteins that bind to the E7 proteins expressed from 17 different HPV types. These studies reveal a number of interactions, some of which are conserved across HPV types and others that are unique to a single HPV species or HPV genus. Binding of E7 to UBR4/p600 is conserved across all virus types, whereas the cellular protein ENC1 binds specifically to the E7s from HPV18 and HPV45, both members of genus alpha, species 7. We identify a specific interaction of HPV16 E7 with ZER1, a substrate specificity factor for a cullin 2 (CUL2)-RING ubiquitin ligase, and show that ZER1 is required for the binding of HPV16 E7 to CUL2. We further show that ZER1 is required for the destabilization of the retinoblastoma tumor suppressor RB1 in HPV16 E7-expressing cells and propose that a CUL2-ZER1 complex functions to target RB1 for degradation in HPV16 E7-expressing cells. These studies refine the current understanding of HPV E7 functions and establish a platform for the rapid identification of virus-host interactions.     

马尔尼菲篮状菌分泌蛋白的鉴定及功能预测分析

目的 鉴定马尔尼菲篮状菌(TM)的分泌蛋白,并初步分析其生物学功能.方法 基于质谱技术对TM菌株(ATCC18224)培养上清进行分泌蛋白组鉴定,利用生物信息学手段预测所鉴定蛋白的亚细胞定位和信号肽剪切位点,以及分析其生物学功能和信号通路.结果 通过2次独立重复实验,共鉴定得到21个分泌蛋白;功能注释结果显示分泌蛋白主...