Introduction of a More Glutaredoxin-like Active Site to PDI Results in Competition between Protein Substrate and Glutathione Binding.

Proteins in the thioredoxin superfamily share a similar fold, contain a -CXXC- active site, and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. Protein disulfide isomerase (PDI) has two -CGHC- active sites. For in vitro studies, oxidation/reduction of PDI during the catalytic cycle is accomplished with glutathione. Glutathione may act as electron donor/acceptor for PDI also in vivo, but at least for oxidation reactions, GSSG probably is not the major electron acceptor and PDI may not have evolved to react with glutathione with high affinity, but merely having adequate affinity for both glutathione and folding proteins/peptides. Glutaredoxins, on the other hand, have a high affinity for glutathione. They commonly have -CXFC- or -CXYC- active site, where the tyrosine residue forms part of the GSH binding groove. Mutating the active site of PDI to a more glutaredoxin-like motif increased its reactivity with glutathione. All such variants showed an increased rate in GSH-dependent reduction or GSSG-dependent oxidation of the active site, as well as a decreased rate of the native disulfide bond formation, with the magnitude of the effect increasing with glutathione concentration. This suggests that these variants lead to competition in binding between glutathione and folding protein substrates.

Increased Heparanase Levels in Urine during Acute Puumala Orthohantavirus Infection Are Associated with Disease Severity.

Old-world orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS), characterized by acute kidney injury (AKI) with transient proteinuria. It seems plausible that proteinuria during acute HFRS is mediated by the disruption of the glomerular filtration barrier (GFB) due to vascular leakage, a hallmark of orthohantavirus-caused diseases. However, direct infection of endothelial cells by orthohantaviruses does not result in increased endothelial permeability, and alternative explanations for vascular leakage and diminished GFB function are necessary. Vascular integrity is partly dependent on an intact endothelial glycocalyx, which is susceptible to cleavage by heparanase (HPSE). To understand the role of glycocalyx degradation in HFRS-associated proteinuria, we investigated the levels of HPSE in urine and plasma during acute, convalescent and recovery stages of HFRS caused by Puumala orthohantavirus. HPSE levels in urine during acute HFRS were significantly increased and strongly associated with the severity of AKI and other markers of disease severity. Furthermore, increased expression of HPSE was detected in vitro in orthohantavirus-infected podocytes, which line the outer surfaces of glomerular capillaries. Taken together, these findings suggest the local activation of HPSE in the kidneys of orthohantavirus-infected patients with the potential to disrupt the endothelial glycocalyx, leading to increased protein leakage through the GFB, resulting in high amounts of proteinuria.

HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses.

Infections with the nine human herpesviruses (HHVs) are globally prevalent and characterized by lifelong persistence. Reactivations can potentially manifest as life-threatening conditions for which the demonstration of viral DNA is essential. In the present study, we developed HERQ-9, a pan-HHV quantitative PCR designed in triplex reactions to differentiate and quantify each of the HHV-DNAs: (i) herpes simplex viruses 1 and 2 and varicella-zoster virus; (ii) Epstein-Barr virus, human cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus; and (iii) HHV-6A, -6B, and -7. The method was validated with prequantified reference standards as well as with mucocutaneous swabs and cerebrospinal fluid, plasma, and tonsillar tissue samples. Our findings highlight the value of multiplexing in the diagnosis of many unsuspected, yet clinically relevant, herpesviruses. In addition, we report here frequent HHV-DNA co-occurrences in clinical samples, including some previously unknown. HERQ-9 exhibited high specificity and sensitivity (LOD95s of ∼10 to ∼17 copies/reaction), with a dynamic range of 101 to 106 copies/µl. Moreover, it performed accurately in the coamplification of both high- and low-abundance targets in the same reaction. In conclusion, we demonstrated that HERQ-9 is suitable for the diagnosis of a plethora of herpesvirus-related diseases. Besides its significance to clinical management, the method is valuable for the assessment of hitherto-unexplored synergistic effects of herpesvirus coinfections. Furthermore, its high sensitivity enables studies on the human virome, often dealing with minute quantities of persisting HHVs.IMPORTANCE By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.

MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction.

MC159 is a viral FLIP (FLICE inhibitory protein) encoded by the molluscum contagiosum virus (MCV) enabling MCV to evade antiviral immunity and to establish persistent infections in humans. Here, we show that MC159 contains a functional SH3 binding motif, which mediates avid and selective binding to SH3BP4, a signaling protein known to regulate endocytic trafficking and suppress cellular autophagy. The capacity to bind SH3BP4 was dispensable for regulation of NF-κB-mediated transcription and suppression of proapoptotic caspase activation but contributed to inhibition of amino acid starvation-induced autophagy by MC159. These results provide new insights into the cellular functions of MC159 and reveal SH3BP4 as a novel host cell factor targeted by a viral immune evasion protein.IMPORTANCE After the eradication of smallpox, molluscum contagiosum virus (MCV) is the only poxvirus restricted to infecting humans. MCV infection is common and causes benign skin lesions that usually resolve spontaneously but may persist for years and grow large, especially in immunocompromised individuals. While not life threatening, MCV infections pose a significant global health burden. No vaccine or specific anti-MCV therapy is available. MCV encodes several proteins that enable it to evade antiviral immunity, a notable example of which is the MC159 protein. In this study, we describe a novel mechanism of action for MC159 involving hijacking of a host cell protein called SH3BP4 to suppress autophagy, a cellular recycling mechanism important for antiviral immunity. This study contributes to our understanding of the host cell interactions of MCV and the molecular function of MC159.

Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands.

The Src Homology-3 (SH3) domains are ubiquitous protein modules that mediate important intracellular protein interactions via binding to short proline-rich consensus motifs in their target proteins. The affinity and specificity of such core SH3 - ligand contacts are typically modest, but additional binding interfaces can give rise to stronger and more specific SH3-mediated interactions. To understand how commonly such robust SH3 interactions occur in the human protein interactome, and to identify these in an unbiased manner we have expressed 324 predicted human SH3 ligands as full-length proteins in mammalian cells, and screened for their preferred SH3 partners using a phage display-based approach. This discovery platform contains an essentially complete repertoire of the ∼300 human SH3 domains, and involves an inherent binding threshold that ensures selective identification of only SH3 interactions with relatively high affinity. Such strong and selective SH3 partners could be identified for only 19 of these 324 predicted ligand proteins, suggesting that the majority of human SH3 interactions are relatively weak, and thereby have capacity for only modest inherent selectivity. The panel of exceptionally robust SH3 interactions identified here provides a rich source of leads and hypotheses for further studies. However, a truly comprehensive characterization of the human SH3 interactome will require novel high-throughput methods based on function instead of absolute binding affinity.

Reorganization of the host cell Crk(L)-PI3 kinase signaling complex by the influenza A virus NS1 protein.

The non-structural protein-1 (NS1) of influenza A virus binds the p85ß subunit of phosphoinositide 3-kinase (PI3K) to induce PI3K activity in the infected cells. Some virus strains encode NS1 containing a motif that binds tightly to the SH3 domain of the cellular adapter proteins Crk and CrkL to potentiate NS1-induced PI3K activation. Here we show that this potentiation involves reorganization of the natural CrkL-p85ß complex into a novel trimeric complex where NS1 serves as a bridging factor. Of note, NS1 proteins that lack the SH3 binding capacity can also associate with CrkL, but in a less stable trimeric complex mediated by p85ß. The data presented here establish Crk proteins as general host cell cofactors of NS1, and show that the enhanced PI3K activation by SH3 binding-competent NS1 variants is mediated by a more efficient tethering of Crk proteins to the NS1-PI3K complex.

The multifunctional enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) converts cysteine-indole-3-acetonitrile to camalexin in the indole-3-acetonitrile metabolic network of Arabidopsis thaliana.

Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, gamma-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate-treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.

Metabolome analysis of biosynthetic mutants reveals a diversity of metabolic changes and allows identification of a large number of new compounds in Arabidopsis.

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.