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Background Infectious mononucleosis (IM) is a contagious illness most frequently caused by Epstein-Barr virus (EBV). There are no recent published studies characterizing the burden of IM in the general US population over time. Methods Nationally representative samples of US ambulatory medical care data were utilized to estimate the N and proportion of visits with an IM diagnosis (based on ICD-9/10 codes) by time period. The National Ambulatory Medical Care Survey (NAMCS: 2006–2015) and National Hospital Ambulatory Care Survey (NHAMCS: 2006–2019) allow estimation of annual visits in free-standing and hospital-based ambulatory care, including emergency departments (EDs). Restricted cubic spline models were implemented to assess temporal trends. NHAMCS data were divided based on availability: combined outpatient and ED (2006–2011) and ED-only (2012–2019). Analyses were conducted using SAS 9.4 & SUDAAN 11.0.3. Results There were 3,182,082 IM-related visits in NAMCS, 599,837 in combined outpatient and ED NHAMCS, and 449,000 in ED-only NHAMCS across study years. For overlapping study years, 2006–2011, across all databases the average annual number of IM-related visits was 419,927. Persons aged 10–30 years comprised the majority of IM-related visits: 61% in NAMCS, 88% in combined NHAMCS, and 85% in ED-only NHAMCS. While 66% of IM visits were made by females in NAMCS, this proportion was lower in combined NHAMCS (58%) and ED-only NHAMCS (51%). Most IM-related visits were among non-Hispanic white patients: 92% in NAMCS, 87% in combined NHAMCS, and 67% in ED-only NHAMCS. Across databases and years, there were no consistent linear trends in IM-related visits (Figure 1). Rates of IM-related visits in NAMCS decreased from 4.18 (95% confidence interval [CI]: 3.02–5.78) in 2006–2009 to 2.89 (95% CI: 1.40–5.96) per 10,000 visits in 2013–2015; decreased from 4.94 (95% CI: 3.41, 7.16) in 2006–2007 to 3.56 (95% CI: 2.33–5.45) per 10,000 visits in 2010–2011 in combined NHAMCS data; and increased from 3.61 (95% CI: 2.30–5.67) in 2012–2014 to 5.98 (95% CI: 3.20–11.17) per 10,000 visits in 2018–2019 in ED-only data. Conclusion This study contributes epidemiologic information concerning IM and EBV by estimating US ambulatory medical care burden of IM over time and describing the demographics of those affected. Disclosures Christina Chandra, MPH, Moderna, Inc.: Contractor Jianheng Li, MBBS, MPH, Moderna, Inc.: Contractor W. Dana Flanders, MD, DSc, MPH, MA, Moderna, Inc.: Contractor Roderick McPhee, MD, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Robert Paris, MD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Philip Buck, PhD, MPH, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Sumana Chandramouli, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Susan Hall, PhD, MS, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Cathy Lally, MSPH, Moderna, Inc.: Contractor.

Children with sickle cell disease (SCD) suffer life-threatening transient aplastic crisis (TAC) when infected with parvovirus B19. In utero, infection of healthy fetuses may result in anemia, hydrops, and death. Unfortunately, although promising vaccine candidates exist, no product has yet been licensed. One barrier to vaccine development has been the lack of a cost-effective, standardized parvovirus B19 neutralization assay. To fill this void, we evaluated the unique region of VP1 (VP1u), which contains prominent targets of neutralizing antibodies. We discovered an antigenic cross-reactivity between VP1 and VP2 that, at first, thwarted the development of a surrogate neutralization assay. We overcame the cross-reactivity by designing a mutated VP1u (VP1uAT) fragment. A new VP1uAT ELISA yielded results well correlated with neutralization (Spearman’s correlation coefficient = 0.581, p = 0.001), superior to results from a standard clinical diagnostic ELISA or an ELISA with virus-like particles. Virus-specific antibodies from children with TAC, measured by the VP1uAT and neutralization assays, but not other assays, gradually increased from days 0 to 120 post-hospitalization. We propose that this novel and technically simple VP1uAT ELISA might now serve as a surrogate for the neutralization assay to support rapid development of a parvovirus B19 vaccine.

Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens.

Respiratory syncytial virus (RSV) is a global public health burden for which no licensed vaccine exists. To aid vaccine development via increased understanding of the protective antibody response to RSV prefusion glycoprotein F (PreF), we performed structural and functional studies using the human neutralizing antibody (nAb) RSB1. The crystal structure of PreF complexed with RSB1 reveals a conformational, pre-fusion specific site V epitope with a unique cross-protomer binding mechanism. We identify shared structural features between nAbs RSB1 and CR9501, elucidating for the first time how diverse germlines obtained from different subjects can develop convergent molecular mechanisms for recognition of the same PreF site of vulnerability. Importantly, RSB1-like nAbs were induced upon immunization with PreF in naturally-primed cattle. Together, this work reveals new details underlying the immunogenicity of site V and further supports PreF-based vaccine development efforts., Author summary Respiratory syncytial virus (RSV) is a persistent, contagious seasonal pathogen and a serious public health threat. While infants are the most at-risk population, with infections potentially leading to bronchiolitis, adults, especially the elderly, are also burdened by RSV-induced respiratory infections. The only treatment currently available for RSV is passive immunization for high-risk infants. Thus, there is a critical need to develop a vaccine for the vast majority of the vulnerable population for which there is no preventative treatment. The RSV fusion protein in its prefusion form (PreF) is the target of the majority of naturally-induced neutralizing antibodies, and several clinical trials are currently evaluating PreF as a promising vaccine candidate. In this study, we solved the X-ray structure of PreF bound to the Fab fragment of a human neutralizing antibody. The structure reveals plasticity of the epitope, as well as a unique molecular signature for antibodies elicited towards this region of PreF. We also find that similar antibodies are induced upon immunization of naturally-primed cattle with a PreF vaccine antigen, suggesting that this epitope is highly immunogenic. These results will help us better understand the human immune response to RSV infection and vaccination, and guide future vaccine-design efforts.

Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site O and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety.

The β-herpesvirus human cytomegalovirus (HCMV) is the leading viral cause of neonatal developmental disabilities. In HCMV, the conserved herpesvirus glycoprotein B (gB) mediates membrane fusion between the viral and host cell membranes, whereas the trimeric gH/gL/gO or the pentameric gH/gL/UL128/UL130/UL31A complexes (Pentamer) bind to cell-specific receptors and provide the triggering signal to gB. Recent structural and functional studies have provided new insights into Pentamer structure, conformational flexibility, location of epitopes for neutralizing antibodies and potential binding sites for cell surface receptors. Together, these data suggest a model where receptor binding triggers a conformational change in Pentamer, allowing it to interact with gB and initiate the membrane fusion process.

Human respiratory syncytial virus (hRSV) is responsible for serious lower respiratory tract disease in infants and in older adults, and remains an important vaccine need. RSV fusion (F) glycoprotein is a key target for neutralizing antibodies. RSV F stabilized in its pre-fusion conformation (DS-Cav1 F) induces high neutralizing antibody titers in naïve animals, but it remains unknown to what extent pre-fusion F can boost pre-existing neutralizing responses in RSV seropositive adults. We here assess DS-Cav1 F immunogenicity in seropositive cattle pre-exposed to bovine RSV, a virus closely related to hRSV. A single immunization with non-adjuvanted DS-Cav1 F strongly boosts RSV neutralizing responses, directed towards pre-fusion F-specific epitopes, whereas a post-fusion F is unable to do so. Vaccination with pre-fusion F thus represents a promising strategy for maternal immunization and for other RSV vaccine target populations such as older adults., Boosting of maternal antibodies against RSV is a potential approach to protect infants from severe infection, but data from animal models is missing. Here Steff et al. test two different boosts in seropositive cattle pre-exposed to bovine RSV and show that pre-fusion F strongly boosts neutralizing responses.

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and organ transplant rejection. The HCMV gH/gL/UL128/UL130/UL131A complex (Pentamer) is the main target of humoral responses and thus a key vaccine candidate. We report two structures of Pentamer bound to human neutralizing antibodies, 8I21 and 9I6, at 3.0 and 5.9 A resolution, respectively. The HCMV gH/gL architecture is similar to that of Epstein-Barr virus (EBV) except for amino-terminal extensions on both subunits. The extension of gL forms a subdomain composed of a three-helix bundle and a β hairpin that acts as a docking site for UL128/UL130/UL131A. Structural analysis reveals that Pentamer is a flexible molecule, and suggests sites for engineering stabilizing mutations. We also identify immunogenic surfaces important for cellular interactions by epitope mapping and functional assays. These results can guide the development of effective vaccines and immunotherapeutics against HCMV.

Parvovirus B19 infections are typically mild in healthy individuals, but can be life threatening in individuals with sickle cell disease (SCD). A Saccharomyces cerevisiae-derived B19 VLP vaccine, now in pre-clinical development, is immunogenic in wild type mice when administered with the adjuvant MF59®. Because SCD alters the immune response, we evaluated the efficacy of this vaccine in a mouse model for SCD. Vaccinated mice with SCD demonstrated similar binding and neutralizing antibody responses to those of heterozygous littermate controls following a prime-boost-boost regimen. Due to the lack of a mouse parvovirus B19 challenge model, we employed a natural mouse pathogen, Sendai virus, to evaluate SCD respiratory tract responses to infection. Normal mucosal and systemic antibody responses were observed in these mice. Results demonstrate that mice with SCD can respond to a VLP vaccine and to a respiratory virus challenge, encouraging rapid development of the B19 vaccine for patients with SCD.

Parvovirus B19 is the causative agent of fifth disease in children, aplastic crisis in those with blood dyscrasias, and hydrops fetalis. Previous parvovirus B19 virus-like-particle (VLP) vaccine candidates were produced by co-infection of insect cells with two baculoviruses, one expressing wild-type VP1 and the other expressing VP2. In humans, the VLPs were immunogenic but reactogenic. We have developed new VLP-based parvovirus B19 vaccine candidates, produced by co-expressing VP2 and either wild-type VP1 or phospholipase-negative VP1 in a regulated ratio from a single plasmid in Saccharomyces cerevisiae. These VLPs are expressed efficiently, are very homogeneous, and can be highly purified. Although VP2 alone can form VLPs, in mouse immunizations, VP1 and the adjuvant MF59 are required to elicit a neutralizing response. Wild-type VLPs and those with phospholipase-negative VP1 are equivalently potent. The purity, homogeneity, yeast origin, and lack of phospholipase activity of these VLPs address potential causes of previously observed reactogenicity.

SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway.

Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV., PLoS Pathogens, 11 (10), ISSN:1553-7374, ISSN:1553-7366

Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection., Cytomegalovirus is a danger to individuals with compromised immune systems and neonates infected in utero. Here the authors show the structure of a neutralizing antibody-bound viral fusion protein glycoprotein B, supporting the development of therapeutic antibodies and vaccines.

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.

Since the discovery of the prototypical Sprouty (Spry) protein in Drosophila, there has been an effort to determine how these novel modulators of the Ras/MAP-kinase pathway function. A clue to their mechanism of action comes from the several highly conserved sequences within all the currently known Spry isoforms: an ∼110-residue cysteine-rich sequence in the C-terminal half that directs Spry proteins to a concentration of signaling proteins at the plasma membrane; a small motif surrounding a tyrosine residue(Y55 in human Spry2) that is responsible for interaction with other proteins. In cultured mammalian cells, hSpry2 inhibits epidermal growth factor receptor(EGFR) endocytosis and subsequently sustains the activation of MAP kinase but negatively regulates the same pathway following stimulation of fibroblast growth factor receptors (FGFRs). Current evidence indicates that Cbl is a key protein that interacts directly with Spry2 following activation of receptor tyrosine kinases (RTKs). It appears to be the ability of Cbl to interact as an E3 ubiquitin ligase on specific target proteins and as a docking protein in other contexts that dictates the differential effects Spry2 has on the Ras/MAP-kinase pathway following EGFR and FGFR activation.

Sprouty (Spry) proteins have been revealed as inhibitors of the Ras/mitogen-activated protein kinase (MAPK) cascade, a pathway crucial for developmental processes initiated by activation of various receptor tyrosine kinases. In COS-1 and Swiss 3T3 cells, all Spry isoforms translocate to the plasma membrane, notably ruffles, following activation. Here we show that microinjection of active Rac induced the translocation of Spry isoforms, indicating that the target of the Spry translocation domain (SpryTD) is downstream of active Rac. Targeted disruption of actin polymerization revealed that the SpryTD target appeared upstream of cytoskeletal rearrangements. Accumulated evidence indicated that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is the likely SpryTD target. Human Spry2TD (hSpry2TD) binds to PtdIns(4,5)P(2) in vesicle-binding assays. hSpry2TD colocalizes with the pleckstrin homology domain of phospholipase Cdelta, which binds PtdIns(4,5)P(2). The plasma membrane localization of hSpry2TD was abolished in ionomycin-treated MDCK cells or when PtdIns(4,5)P(2) was specifically dephosphorylated by overexpression of an engineered, green fluorescent protein-tagged inositol 5-phosphatase. Similarly, Spred, a novel Ras/MAPK inhibitor recently found to contain the conserved cysteine-rich SpryTD, also translocated to peripheral membranes and bound to PtdIns(4,5)P(2). Alignment of the Spry and Spred proteins led us to identify a translocation-defective point mutant, hSpry2 D252. Targeting of hSpry2 to PtdIns(4,5)P(2) was shown to be essential for the down-regulation of Ras/MAPK signaling.

Sickle cell disease (SCD) is caused by a mutant β-globin gene and affects more than 100,000 individuals in the USA alone. Sickled RBCs and ischemic events cause tissue damage, altered splenic architecture, and inflammation. When children are infected with parvovirus B19, they can develop life-threatening aplastic crisis. Previous reports have described immune defects among patients with SCD, and their potential correlations with splenic dysfunction or inflammation. In a multi-center study involving St. Jude, NIH, Novartis, and GlaxoSmithKline, we examined (i) inflammatory cytokines/chemokines in patients with SCD, treated or untreated with hydroxyurea (HU); (ii) immune responses toward parvovirus B19 infections in children with SCD; and (iii) immune responses in a mouse model for SCD against a candidate Saccharomyces cerevisiae-derived parvovirus B19 VLP vaccine. Results showed that HU treatment reduced serum cytokines/chemokines in patients with SCD (e.g. TGFα, TNFα, and sVCAM-1). Both HU-treated and untreated children, regardless of their cytokine/chemokine profile, responded to parvovirus B19. Binding antibodies were apparent in the nasal cavity (a common point-of entry for the pathogen) and sera by day 7 post-infection, and virus-specific neutralizing antibodies were induced. In transgenic mice with SCD (expressing human α, βS, and γ globin genes), we observed normal antibody activities toward the VLP vaccine. Together, data show that parvovirus B19 -specific B cells withstand the aberrant SCD environment better than their previously-described Streptococcus pneumonia-specific B cell counterparts. Data encourage rapid development of the VLP vaccine to prevent aplastic crises in children with SCD.

The Sprouty (Spry) proteins function as inhibitors of the Ras-ERK pathway downstream of various receptor tyrosine kinases. In this study, we have identified Tesk1 (testicular protein kinase 1) as a novel regulator of Spry2 function. Endogenous Tesk1 and Spry2 exist in a complex in cell lines and mouse tissues. Tesk1 coexpression relocalizes Spry2 to vesicles including endosomes, inhibiting its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, Tesk1 binding leads to a loss of Spry2 function as an inhibitor of ERK phosphorylation and reverses inhibition of basic fibroblast growth factor (bFGF)- and nerve growth factor-induced neurite outgrowth in PC12 cells by Spry2. Furthermore, depletion of endogenous Tesk1 in PC12 cells leads to a reduction in neurite outgrowth induced by bFGF. Tesk1 nullifies the inhibitory effect of Spry2 by abrogating its interaction with the adaptor protein Grb2 and interfering with its serine dephosphorylation upon bFGF and FGF receptor 1 stimulation by impeding its binding to the catalytic subunit of protein phosphatase 2A. A construct of Tesk1 that binds to Spry2 but does not localize to the vesicles does not interfere with its function, highlighting the importance of subcellular localization of Tesk1 in this context. Conversely, Tesk1 does not affect interaction of Spry2 with the E3 ubiquitin ligase, c-Cbl, and consequently, does not affect its inhibition of Cbl-mediated ubiquitination of the epidermal growth factor receptor. By selectively modulating the downstream effects of Spry2, Tesk1 may thus serve as a molecular determinant of the signaling outcome.

In the context of fibroblast growth factor (FGF) signaling, Sprouty2 (Spry2) is the most profound inhibitor of the Ras/ERK pathway as compared with other Spry isoforms. An exclusive, necessary, but cryptic PXXPXR motif in the C terminus of Spry2 is revealed upon stimulation. The activation of Spry2 appears to be linked to sequences in the N-terminal half of the protein and correlated with a bandshifting seen on SDS-PAGE. The band-shifting is likely caused by changes in the phosphorylation status of key Ser and Thr residues following receptor stimulation. Dephosphorylation of at least two conserved Ser residues (Ser-112 and Ser-115) within a conserved Ser/Thr sequence is accomplished upon stimulation by a phosphatase that binds to Spry2 around residues 50-60. We show that human Spry2 co-immunoprecipitates with both the catalytic and the regulatory subunits of protein phosphatase 2A (PP2A-C and PP2A-A, respectively) in cells upon FGF receptor (FGFR) activation. PP2A-A binds directly to Spry2, but not to Spry2Delta50-60 (Delta50-60), and the activity of PP2A increases with both FGF treatment and FGFR1 overexpression. c-Cbl and PP2A-A compete for binding centered around Tyr-55 on Spry2. We show that there are at least two distinct pools of Spry2, one that binds PP2A and another that binds c-Cbl. c-Cbl binding likely targets Spry2 for ubiquitin-linked destruction, whereas the phosphatase binding and activity are necessary to dephosphorylate specific Ser/Thr residues. The resulting change in tertiary structure enables the Pro-rich motif to be revealed with subsequent binding of Grb2, a necessary step for Spry2 to act as a Ras/ERK pathway inhibitor in FGF signaling.

Because the Sprouty (Spry) proteins were shown to be inhibitors of the mainstream Ras/ERK pathway, there has been considerable interest in ascertaining their mechanism of action especially since a possible role as tumor suppressors for these inhibitory proteins has been suggested. We compared the ability of the mammalian Spry isoforms to inhibit the Ras/ERK pathway in the context of fibroblast growth factor receptor (FGFR) signaling. Spry2 is considerably more inhibitory than Spry1 or Spry4, and this correlates with the binding to Grb2 via a C-terminal proline-rich sequence that is found exclusively on Spry2. This PXXPXR motif binds directly to the N-terminal Src homology domain 3 of Grb2, and when added onto the C terminus of Spry4 the resultant chimera inhibits the Ras/ERK pathway. The ability to inhibit neurite outgrowth in PC-12 cells correlates with the propensity of Spry isoforms and engineered constructs to inhibit the phosphorylation of ERK1/2. The PXXPXR motif is cryptic in unstimulated cells, and it is postulated that Spry2 undergoes a conformational change following FGFR stimulation, enabling the subsequent interaction with Grb2. We present evidence that Spry2 can compete with the RasGEF (guanine nucleotide exchange factor) SOS1 for binding to Grb2, resulting in the inhibition of phosphorylation of ERK1/2.

Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl-CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS-1, from the banana fruit is reported. MaMS-1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS-1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS-1 also shared an extensive sequence homology (79-81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS-1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS-1 expression in FG fruit could be stimulated by treatment with 1 microl l(-1) exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene-inducible.