A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry.

Glycosylation is a common PTM of plant proteins that impacts a large number of important biological processes. Nevertheless, the impacts of differential site occupancy and the nature of specific glycoforms are obscure. Historically, characterization of glycoproteins has been difficult due to the distinct physicochemical properties of the peptidyl and glycan moieties, the variable and dynamic nature of the glycosylation process, their heterogeneous nature, and the low relative abundance of each glycoform. In this study, we explore a new pipeline developed for large-scale empirical identification of N-linked glycoproteins of tomato fruit as part of our ongoing efforts to characterize the tomato secretome. The workflow presented involves a combination of lectin affinity, tryptic digestion, ion-pairing HILIC, and precursor ion-driven data-dependent MS/MS analysis with a script to facilitate the identification and characterization of occupied N-linked glycosylation sites. A total of 212 glycoproteins were identified in this study, in which 26 glycopeptides from 24 glycoproteins were successfully characterized in just one HILIC fraction. Further precursor ion discovery-based MS/MS and deglycosylation followed by high accuracy and resolution MS analysis were used to confirm the glycosylation sites and determine site occupancy rates. The workflow reported is robust and capable of producing large amounts of empirical data involving N-linked glycosylation sites and their associated glycoforms.

c-Cbl tyrosine kinase-binding domain mutant G306E abolishes the interaction of c-Cbl with CD38 and fails to promote retinoic acid-induced cell differentiation and G0 arrest.

Retinoic acid (RA) causes HL-60 human myeloblastic leukemia cell myeloid differentiation that is dependent on MAPK signaling. The process is propelled by c-Cbl, which binds the CD38 receptor as part of a signaling complex generating MAPK signaling. Here we report that the capability of c-Cbl to do this is lost in the G306E tyrosine kinase-binding domain mutant. Unlike wild-type (WT) c-Cbl, the G306E mutant c-Cbl fails to propel RA-induced differentiation, and disrupts the normal association with CD38. The G306E mutant does, like WT c-Cbl, co-immunoprecipitate with Vav, Slp-76, and p38. But unlike WT c-Cbl, does not cause MAPK signaling. In contrast, the C381A Ring finger domain mutant functions like WT c-Cbl. It binds CD38 and is part of the same apparent c-Cbl/Slp-76/Vav/p38 signaling complex. The C381A mutant causes MAPK signaling and propels RA-induced differentiation. In addition to HL-60 cells and their WT or mutant c-Cbl stable transfectants, the c-Cbl/Vav/Slp-76 complex is also found in NB4 cells where c-Cbl was previously also found to bind CD38. The data are consistent with a model in which the G306E mutant c-Cbl forms a signaling complex that includes Slp-76, Vav, and p38; but does not drive MAPK signaling because it fails to bind the CD38 receptor. Without the G306E mutation the c-Cbl unites CD38 with the signaling complex and delivers a MAPK signal that drives RA-induced differentiation. The results demonstrate the importance of the Gly306 residue in the ability of c-Cbl to propel RA-induced differentiation.

LC-MS/MS quantification of asymmetric dimethyl arginine and symmetric dimethyl arginine in plasma using surrogate matrix and derivatization with fluorescamine.

Aim: A novel LC-MS/MS method using a surrogate matrix and derivatization with fluorescamine was developed and validated for simultaneous quantification of asymmetric dimethyl arginine and symmetric dimethyl arginine. Methods & results: Asymmetric dimethyl arginine, symmetric dimethyl arginine and corresponding internal standards were extracted using protein precipitation and derivatization with fluorescamine followed by SPE. Derivatives were analyzed by turbo ion spray LC-MS/MS in the positive ion mode. Methodology was successfully transferred across multiple preclinical species and utilized in the support of several investigative studies. Conclusion: A new LC-MS/MS analytical methodology that utilizes a surrogate matrix and derivatization with fluorescamine was successfully developed and validated.

Nicotinamide cooperates with retinoic acid and 1,25-dihydroxyvitamin D(3) to regulate cell differentiation and cell cycle arrest of human myeloblastic leukemia cells.

Nicotinamide, the amide derivative of vitamin B(3), cooperates with retinoic acid (RA), a form of vitamin A, and 1,25-dihydroxyvitamin D(3) (D3), to regulate cell differentiation and proliferation of human myeloblastic leukemia cells. In human myeloblastic leukemia cells, RA or D3 are known to cause MAPK signaling leading to myeloid or monocytic differentiation and G0 cell cycle arrest. In this process, RA or D3 induces the early expression of CD38, a receptor that causes ERK signaling and propels further differentiation. Our study demonstrates that nicotinamide in combination with RA or D3 affected induced expression levels of CD38, CD11b and CD14, suggesting a cooperative function of nicotinamide and RA or D3. Nicotinamide transiently retarded the initial RA- or D3-induced expression of CD38, which subsequently reached the same nearly 100% expression. Nicotinamide induced ERK activation and further enhanced the RA-induced ERK activation, but the D3-induced ERK activation was diminished by nicotinamide, although levels still exceeded those induced by RA, suggesting lineage-specific nicotinamide responses. Nicotinamide enhanced both RA- and D3-induced CD11b expression, inducible oxidative metabolism, and G0 cell cycle arrest, accelerating their induced occurrence in all instances. Consistent with this, the RA- or D3-induced downregulation of PARP was enhanced by nicotinamide. Nicotinamide thus regulated RA- or D3-induced differentiation and G0 arrest, causing a transient delay in certain early aspects of the progression to terminal differentiation but ultimately accelerating the occurrence of terminally, functionally differentiated G0 cells.

Phosphorylation of c-Cbl and p85 PI3K driven by all-trans retinoic acid and CD38 depends on Lyn kinase activity.

The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driven signaling. Cells treated with ATRA for 48h followed by one hour of PP2 incubation show SFK/Lyn kinase inhibition. We observed that Lyn inhibition blocked c-Cbl and p85/p55 PI3K phosphorylation driven by the anti-CD38 agonistic mAb IB4 in ATRA-treated HL-60 cells and untreated CD38+ transfectants. In contrast, cells cultured for 48h following concurrent ATRA and PP2 treatment did not show Lyn inhibition, suggesting ATRA regulates the effects on Lyn. 48h of co-treatment preserved CD38-stimulated c-Cbl and p85/p55 PI3K phosphorylation indicating Lyn kinase activity is necessary for these events. In contrast another SFK inhibitor (dasatinib) which blocks Lyn activity with ATRA co-treatment prevented ATRA-induced c-Cbl phosphorylation and crippled p85 PI3K phosphorylation, indicating Lyn kinase activity is important for ATRA-propelled events potentially regulated by CD38. We found that loss of Lyn activity coincided with a decrease in Vav1/Lyn/CD38 and SLP-76/Lyn/CD38 interaction, suggesting these molecules form a complex that regulates CD38 signaling. Lyn inhibition also reduced Lyn and CD38 binding to p85 PI3K, indicating CD38 facilitates a complex responsible for PI3K phosphorylation. Therefore, Lyn kinase activity is important for CD38-associated signaling that may drive ATRA-induced differentiation.

Leveraging non-targeted metabolite profiling via statistical genomics.

One of the challenges of systems biology is to integrate multiple sources of data in order to build a cohesive view of the system of study. Here we describe the mass spectrometry based profiling of maize kernels, a model system for genomic studies and a cornerstone of the agroeconomy. Using a network analysis, we can include 97.5% of the 8,710 features detected from 210 varieties into a single framework. More conservatively, 47.1% of compounds detected can be organized into a network with 48 distinct modules. Eigenvalues were calculated for each module and then used as inputs for genome-wide association studies. Nineteen modules returned significant results, illustrating the genetic control of biochemical networks within the maize kernel. Our approach leverages the correlations between the genome and metabolome to mutually enhance their annotation and thus enable biological interpretation. This method is applicable to any organism with sufficient bioinformatic resources.

Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells.

All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation.

Arsenic trioxide cooperates with all trans retinoic acid to enhance mitogen-activated protein kinase activation and differentiation in PML-RARalpha negative human myeloblastic leukemia cells.

Arsenic trioxide (ATO) synergistically promotes all trans retinoic acid (ATRA)-induced differentiation of PML-RARalpha negative HL-60 myeloblastic leukemia cells. In PML-RARalpha positive myeloid leukemia cells, ATO is known to cause degradation of PML-RARalpha with subsequent induced myeloid differentiation. We found that ATO by itself does not cause differentiation of the PML-RARalpha negative HL-60 cells, but enhances ATRA's capability to cause differentiation. ATO augmented ATRA-induced RAF/MEK/ERK axis signaling, expression of CD11b and p47(PHOX), and inducible oxidative metabolism. ATO enhanced ATRA-induced population growth retardation without evidence of apoptosis or enhanced G1/G0 growth arrest. Compared to ATRA-treated cells, the ATRA plus ATO-treated cells progressed more slowly through the cell cycle as detected by a slower rate of accumulation in G2/M following nocodazole treatment. Hoechst/PI staining showed that low-dose ATO did not induce apoptosis. In summary, our results indicate that ATO in conjunction with ATRA is of potential chemotherapeutic use in PML-RARalpha negative leukemias.

c-Cbl interacts with CD38 and promotes retinoic acid-induced differentiation and G0 arrest of human myeloblastic leukemia cells.

Retinoic acid (RA) is known to regulate cell growth and differentiation. In HL-60 human myeloblastic leukemia cells, it causes mitogen-activated protein kinase (MAPK) signaling leading to myeloid differentiation and G(0) cell cycle arrest. This communication reports that expression of the Cbl adaptor caused enhanced extracellular signal-regulated kinase 2 activation and promoted RA-induced differentiation and G(0)-arrest. Stable transfectants ectopically expressing c-Cbl underwent myeloid differentiation faster than wild-type (wt) cells when treated with RA. In contrast, c-Cbl knockdown stable transfectants differentiated slower than wt cells when treated with RA. Cells ectopically expressing c-Cbl had enhanced CD38 expression when treated with RA, and cells ectopically expressing CD38 had enhanced c-Cbl expression, even without with RA, suggesting an interaction between c-Cbl and CD38. Fluorescence resource energy transfer and coimmunoprecipitation showed that c-Cbl and CD38 bind each other. RA causes the gradual down-regulation and eventual loss of c-Cbl expression, resulting in loss of the Cbl-CD38 interaction, suggesting that c-Cbl plays a relatively early role in promoting RA-induced differentiation. RA-induced differentiation can thus be propelled by c-Cbl and by CD38, both of which bind together, enhance the expression of each other, and cause MAPK signaling. There thus seems to be a cooperative role for c-Cbl and CD38, reflected in their direct binding, in propulsion of RA-induced differentiation.

Picornavirus genome replication. Identification of the surface of the poliovirus (PV) 3C dimer that interacts with PV 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C2-3Dpol complex.

Picornaviruses have a peptide termed VPg covalently linked to the 5'-end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precursor thereof, is used as a primer by the viral RNA-dependent RNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagenesis of 3C to identify the surface of 3C with which 3Dpol interacts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants when placed in the context of a poliovirus mutant containing 3Dpol-R455A, a residue on the back of the thumb required for VPg uridylylation. These data were used to guide molecular docking of the structures for a poliovirus 3C dimer and 3Dpol, leading to a structural model for the 3C(2)-3Dpol complex that extrapolates well to all picornaviruses.

Human rhinovirus type 14 gain-of-function mutants for oriI utilization define residues of 3C(D) and 3Dpol that contribute to assembly and stability of the picornavirus VPg uridylylation complex.

VPg linkage to the 5' ends of picornavirus RNAs requires production of VPg-pUpU. VPg-pUpU is templated by an RNA stem-loop (the cre or oriI) found at different locations in picornavirus genomes. At least one adaptive mutation is required for human rhinovirus type 14 (HRV-14) to use poliovirus type 3 (PV-3) or PV-1 oriI efficiently. One mutation changes Leu-94 of 3C to Pro; the other changes Asp-406 of 3Dpol to Asn. By using an in vitro VPg uridylylation system for HRV-14 that recapitulates biological phenotypes, we show that the 3C adaptive mutation functions at the level of 3C(D) and the 3D adaptive mutation functions at the level of 3Dpol. Pro-94 3C(D) has an expanded specificity and enhanced stability relative to wild-type 3C(D) that leads to production of more processive uridylylation complexes. PV-1/HRV-14 oriI chimeras reveal sequence specificity in 3C(D) recognition of oriI that resides in the upper stem. Asn-406 3Dpol is as active as wild-type 3Dpol in RNA-primed reactions but exhibits greater VPg uridylylation activity due to more efficient recruitment to and retention in the VPg uridylylation complex. Asn-406 3Dpol from PV-1 exhibits identical behavior. These studies suggest a two-step binding mechanism in the assembly of the 3C(D)-oriI complex that leads to unwinding of at least the upper stem of oriI and provide additional support for a direct interaction between the back of the thumb of 3Dpol and 3C that is required for 3Dpol recruitment to and retention in the uridylylation complex.

Intricate transmission routes and interactions between picorna-like viruses (Kashmir bee virus and sacbrood virus) with the honeybee host and the parasitic varroa mite.

Viral diseases of honeybees are a major problem in apiculture, causing serious economic losses worldwide, especially in combination with varroa mites. To increase understanding of the relationship among viruses, mites and colony decline, the tripartite relationships among bees, viruses [Kashmir bee virus (KBV) and sacbrood virus (SBV)] and varroa mites have been investigated systematically. To develop an antibody-based test for KBV, two structural recombinant proteins were purified for polyclonal-antibody production. By using ELISA and RT-PCR, the presence of KBV and SBV was studied comparatively in different developmental stages and castes of bees. The results demonstrated that KBV may persist as a viral genome with extremely low levels of viral-capsid proteins and that KBV and SBV can co-infect honeybees. This study indicated the presence of KBV and SBV RNAs in both queens and eggs by RT-PCR, suggesting a route of transovarial transmission. Horizontal transmission is also very likely among adult bees and from adult workers to larvae through contaminated food resources, because both viruses have been detected in all developmental stages and food sources (brood food, honey, pollen and royal jelly). Furthermore, it was demonstrated that mites were another possible route of horizontal transmission, as both viruses were detected in mites and their saliva. This study, for the first time, detected co-occurrence of viruses in varroa, further underlining the importance of the mites in vectoring different bee viruses. Therefore, these results indicated that multiple infection routes exist for honeybee viral diseases.

The role of varroa mites in infections of Kashmir bee virus (KBV) and deformed wing virus (DWV) in honey bees.

To determine the roles of varroa mites in activating or vectoring viral infections, we performed quantitative comparison of viral infections between bees with and without mites by dot blot analysis and enzyme-linked immunosorbent assay (ELISA). Under natural and artificial mite infestations, bee pupae contained significantly higher levels of Kashmir bee virus (KBV) and deformed wing virus (DWV) RNAs and KBV structural proteins than mite-free pupae. Moreover, in mite-infested bee pupae, DWV had amplified to extremely high titers with viral genomic RNA being clearly visible after separation of total bee RNA in agarose gels. Linear regression analysis has shown a positive correlation between the number of mites introduced and the levels of viral RNAs. The detection of viral RNAs in the nymph and adult mites underline the possible role of varroa in virus transmission. However, most groups of virus-free adult mites (9/12) were associated with bee pupae heavily infected by viruses, suggesting that the elevated viral titers in mite-infested pupae more likely resulted from activated viral replication. Based on these observations and our concurrent research demonstrating suppressed immune responses in bees infested with mites, we propose that parasitization by varroa suppresses the immunity of honey bees, leading to activation of persistent, latent viral infection.