The Crystal Structure of PPIL1 Bound to Cyclosporine A Suggests a Binding Mode for a Linear Epitope of the SKIP Protein

BACKGROUND: The removal of introns from pre-mRNA is carried out by a large macromolecular machine called the spliceosome. The peptidyl-prolyl cis/trans isomerase PPIL1 is a component of the human spliceosome and binds to the spliceosomal SKIP protein via a binding site distinct from its active site. PRINCIPAL FINDINGS: Here, we have studied the PPIL1 protein and its interaction with SKIP biochemically and by X-ray crystallography. A minimal linear binding epitope derived from the SKIP protein could be determined using a peptide array. A 36-residue region of SKIP centred on an eight-residue epitope suffices to bind PPIL1 in pull-down experiments. The crystal structure of PPIL1 in complex with the inhibitor cyclosporine A (CsA) was obtained at a resolution of 1.15 A and exhibited two bound Cd(2+) ions that enabled SAD phasing. PPIL1 residues that have previously been implicated in binding of SKIP are involved in the coordination of Cd(2+) ions in the present crystal structure. Employing the present crystal structure, the determined minimal binding epitope and previously published NMR data, a molecular docking study was performed. In the docked model of the PPIL1.SKIP interaction, a proline residue of SKIP is buried in a hydrophobic pocket of PPIL1. This hydrophobic contact is encircled by several hydrogen bonds between the SKIP peptide and PPIL1. CONCLUSION: We characterized a short, linear epitope of SKIP that is sufficient to bind the PPIL1 protein. Our data indicate that this SKIP peptide could function in recruiting PPIL1 into the core of the spliceosome. We present a molecular model for the binding mode of SKIP to PPIL1 which emphasizes the versatility of cyclophilin-type PPIases to engage in additional interactions with other proteins apart from active site contacts despite their limited surface area

Architecture of a nascent viral fusion pore

Enveloped viruses use specialized protein machinery to fuse the viral membrane with that of the host cell during cell invasion. In influenza virus, hundreds of copies of the haemagglutinin (HA) fusion glycoprotein project from the virus surface. Despite intensive study of HA and its fusion activity, the protein's modus operandi in manipulating viral and target membranes to catalyse their fusion is poorly understood. Here, the three-dimensional architecture of influenza virus–liposome complexes at pH 5.5 was investigated by electron cryo-tomography. Tomographic reconstructions show that early stages of membrane remodeling take place in a target membrane-centric manner, progressing from punctate dimples, to the formation of a pinched liposomal funnel that may impinge on the apparently unperturbed viral envelope. The results suggest that the M1 matrix layer serves as an endoskeleton for the virus and a foundation for HA during membrane fusion. Fluorescence spectroscopy monitoring fusion between liposomes and virions shows that leakage of liposome contents takes place more rapidly than lipid mixing at pH 5.5. The relation of ‘leaky' fusion to the observed prefusion structures is discussed

The human equilibrative nucleoside transporter 1 mediates in vitro cytarabine sensitivity in childhood acute myeloid leukaemia

Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=−0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=−0.30; P=0.04), decitabine (rp=−0.29; P=0.04) and gemcitabine (rp=−0.33; P=0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (rp=−0.31; P=0.03) and decitabine (rp=0.33; P=0.03), respectively. The dCK/PN-I ratio correlated inversely with LC50 values for gemcitabine (rp=−0.45, P=0.001) and the dCK/CDA ratio seemed to correlate with LC50 values for decitabine (rp=−0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML

A Novel Inactivated Intranasal Respiratory Syncytial Virus Vaccine Promotes Viral Clearance without Th2 Associated Vaccine-Enhanced Disease

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960's led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations.In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines.These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology

An apoplastic peptide signal activates salicylic acid signalling in maize

Control of plant pathogen resistance or susceptibility largely depends on the promotion of either cell survival or cell death. In this context, papain-like cysteine proteases (PLCPs) regulate plant defence to drive cell death and protection against biotrophic pathogens. In maize (Zea mays), PLCPs are crucial in the orchestration of salicylic acid (SA)-dependent defence signalling. Despite this central role in immunity, it remains unknown how PLCPs are activated, and which downstream signals they induce to trigger plant immunity. Here, we present the discovery of an immune signalling peptide, Zea mays immune signalling peptide 1 (Zip1). A mass spectrometry approach identified the Zip1 peptide being produced after salicylic acid (SA) treatment. In vitro studies using recombinant proteins demonstrate that PLCPs are required to release bioactive Zip1 from its propeptide precursor (PROZIP1). Strikingly, Zip1 treatment strongly elicits SA accumulation in maize leaves. Moreover, RNAseq based transcriptome analyses revealed that Zip1 and SA treatments induce highly overlapping transcriptional changes. Consequently, Zip1 promotes the infection of the necrotrophic pathogen Botrytis cinerea in maize, while it reduces virulence of the biotrophic fungus Ustilago maydis. Together, Zip1 represents the previously missing signal that is released by PLCPs to activate SA defence signalling

Mechanisms of Resistance to Decitabine in the Myelodysplastic Syndrome

Purpose: The DNA methylation inhibitor 5-aza-29-deoxycytidine (DAC) is approved for the treatment of myelodysplastic syndromes (MDS), but resistance to DAC develops during treatment and mechanisms of resistance remain unknown. Therefore, we investigated mechanisms of primary and secondary resistance to DAC in MDS. Patients and Methods: We performed Quantitative Real-Time PCR to examine expression of genes related to DAC metabolism prior to therapy in 32 responders and non-responders with MDS as well as 14 patients who achieved a complete remission and subsequently relapsed while on therapy (secondary resistance). We then performed quantitative methylation analyses by bisulfite pyrosequencing of 10 genes as well as Methylated CpG Island Amplification Microarray (MCAM) analysis of global methylation in secondary resistance. Results: Most genes showed no differences by response, but the CDA/DCK ratio was 3 fold higher in non-responders than responders (P,.05), suggesting that this could be a mechanism of primary resistance. There were no significant differences at relapse in DAC metabolism genes, and no DCK mutations were detected. Global methylation measured by the LINE1 assay was lower at relapse than at diagnosis (P,.05). On average, the methylation of 10 genes was lower at relapse (16.1%) compared to diagnosis (18.1%) (P,.05).MCAM analysis showed decreased methylation of an average of 4.5 % (range 0.6%– 9.7%) of the genes at relapse. By contrast, new cytogenetic changes were found in 20 % of patients

Dengue Virus Ensures Its Fusion in Late Endosomes Using Compartment-Specific Lipids

Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues