Detection of HPV E6 oncoprotein from urine via a novel immunochromatographic assay.

Cervical cancer is a significant public health problem, especially in low- and middle-income countries, where women have little access to cervical cancer screening; consequently 80% of cervical cancer related mortality occurs in these regions. The development of screening methods that need less infrastructure thus represents an urgent medical need. The study aims to compare the detection rates of high-risk human papillomavirus 16 and 18 E6 oncoprotein in urine, vaginal self-collected, and cervical scrapes of women using the OncoE6™ Cervical Test and compare the HPV16 and/or HPV18 E6 detection rates with the HPV DNA testing. Paired urine, vaginal self-collected and cervical specimens were collected from 124 women who participated in cervical cancer screening or treatment in this proof-of-concept study and underwent to HPV16/18-E6 testing and high-risk HPV DNA testing prior to treatment of cervical neoplasia or cancer. Concordance between urinary, vaginal and cervical HPV16/18-E6 and HPV-DNA testing was evaluated for patients classified as negative group (

DM influences the abundance of major histocompatibility complex class II alleles with low affinity for class II-associated invariant chain peptides via multiple mechanisms.

DM catalyses class II-associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation-sensitive anti-class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse-chase experiments indicates that DM increases the half-life of affected alleles. Unexpectedly, the pulse-chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM-dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide-receptive DR0404 molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM-mediated increases in the abundance of low-CLIP-affinity alleles.

Posttranslational regulation of I-Ed by affinity for CLIP.

Several MHC class II alleles linked with autoimmune diseases form unusually low stability complexes with CLIP, leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. To investigate cellular consequences of altering class II/CLIP affinity, we evaluated invariant chain (Ii) mutants with varying CLIP affinity for a mouse class II allele, I-E(d), which has low affinity for wild-type CLIP and is associated with a mouse model of spontaneous, autoimmune joint inflammation. Increasing CLIP affinity for I-E(d) resulted in increased cell surface and total cellular abundance and half-life of I-E(d). This reveals a post-endoplasmic reticulum chaperoning capacity of Ii via its CLIP peptides. Quantitative effects on I-E(d) were less pronounced in DM-expressing cells, suggesting complementary chaperoning effects mediated by Ii and DM, and implying that the impact of allelic variation in CLIP affinity on immune responses will be highest in cells with limited DM activity. Differences in the ability of cell lines expressing wild-type or high-CLIP-affinity mutant Ii to present Ag to T cells suggest a model in which increased CLIP affinity for class II serves to restrict peptide loading to DM-containing compartments, ensuring proper editing of antigenic peptides.

PDZ protein interactions underlying NMDA receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors.

In neuronal synapses, PDZ domains [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] of PSD-95 proteins interact with C termini of NMDA receptor [NMDAR (NR)] subunits, linking them to downstream neurotoxic signaling molecules. Perturbing NMDAR/PSD-95 interactions with a Tat peptide comprising the nine C-terminal residues of the NR2B subunit (Tat-NR2B9c) reduces neurons' vulnerability to excitotoxicity and ischemia. However, NR subunit C termini may bind many of >240 cellular PDZs, any of which could mediate neurotoxic signaling independently of PSD-95. Here, we performed a proteomic and biochemical analysis of the interactions of all known human PDZs with synaptic signaling proteins including NR1, NR2A-NR2D, and neuronal nitric oxide synthase (nNOS). Tat-NR2B9c, whose interactions define PDZs involved in neurotoxic signaling, was also used. NR2A-NR2D subunits and Tat-NR2B9c had similar, highly specific, PDZ protein interactions, of which the strongest were with the PSD-95 family members (PSD-95, PSD-93, SAP97, and SAP102) and Tax interaction protein 1 (TIP1). The PSD-95 PDZ2 domain bound NR2A-NR2C subunits most strongly (EC50, approximately 1 microM), and fusing the NR2B C terminus to Tat enhanced its affinity for PSD-95 PDZ2 by >100-fold (EC50, approximately 7 nM). IC50 values for Tat-NR2B9c inhibiting NR2A-NR2C/PSD-95 interactions (approximately 1-10 microM) and nNOS/PSD-95 interactions (200 nM) confirmed the feasibility of such inhibition. To determine which of the PDZ interactions of Tat-NR2B9c mediate neuroprotection, one of PSD-95, PSD-93, SAP97, SAP102, TIP1, or nNOS expression was inhibited in cortical neurons exposed to NMDA toxicity. Only neurons lacking PSD-95 or nNOS but not PSD-93, SAP97, SAP102, or TIP1 exhibited reduced excitotoxic vulnerability. Thus, despite the ubiquitousness of PDZ domain-containing proteins, PSD-95 and nNOS above any other PDZ proteins are keys in effecting NMDAR-dependent excitotoxicity. Consequently, PSD-95 inhibition may constitute a highly specific strategy for treating excitotoxic disorders.

Interaction of HLA-DR with an acidic face of HLA-DM disrupts sequence-dependent interactions with peptides.

HLA-DM (DM) edits major histocompatibility complex class II (MHCII)-bound peptides in endocytic compartments and stabilizes empty MHCII molecules. Crystal structures of DM have revealed similarity to MHCII but not how DM and MHCII interact. We used mutagenesis to map a MHCII-interacting surface on DM. Mutations on this surface impair DM action on HLA-DR and -DP in cells and DM-dependent peptide loading in vitro. The orientation of DM and MHCII predicted by these studies guided design of soluble DM and DR molecules fused to leucine zippers via their beta chains, resulting in stable DM/DR complexes. Peptide release from the complexes was fast and only weakly sequence dependent, arguing that DM diminishes the selectivity of the MHCII groove. Analysis of soluble DM action on soluble DR/peptide complexes corroborates this conclusion.

Point mutations in or near the antigen-binding groove of HLA-DR3 implicate class II-associated invariant chain peptide affinity as a constraint on MHC class II polymorphism.

During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II alphabeta dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations betaS11F, betaS13Y, betaQ70R, betaK71E, betaK71N, and betaR74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as alphaG20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.

Formation of two peptide/MHC II isomers is catalyzed differentially by HLA-DM.

Major histocompatability class II proteins are transmembrane alphabeta-heterodimers that present peptides to T-cells. MHC II may bind exogenous peptides directly at the cell surface. Alternatively, peptides derived from processing of endosomal protein may bind to MHC II in endosomal compartments. There, HLA-DM catalyzes the formation of peptide/MHC complexes, which are then transported to the cell surface. Here we report evidence that the peptide Ii CLIP 81-104 binds to DR*0404 in two alternate registries, whose dissociation rates, while kinetically indistinguishable at pH 5.3 and 37 degrees C, are kinetically resolved in the presence of HLA-DM. In one registry isomer, CLIP Met 91 is placed in the N-terminal P1 pocket of DR*0404, and peptide dissociation is readily catalyzed by HLA-DM. In a second proposed registry, likely with CLIP Leu 97 in the P1 pocket, the complex is substantially less sensitive to HLA-DM catalysis. Without HLA-DM, or at pH 7, the fraction of each isomer formed in solution is relatively insensitive to the duration of incubation with peptide. However, with HLA-DM, the fraction of the DM-insensitive isomer is dramatically influenced by peptide incubation time. The mechanism of isomer formation appears to be determined by the HLA-DM-modified relative association to the two registries, followed by HLA-DM-catalyzed dissociation of each isomer and rebinding, leading to a final isomer composition determined by these kinetic constants. Intramolecular isomer interconversion does not appear to be involved. The behavior of these complexes may provide a model for peptide editing by DM in endosomes.

Structural factors contributing to DM susceptibility of MHC class II/peptide complexes.

Peptide loading of MHC class II (MHCII) molecules is assisted by HLA-DM, which releases invariant chain peptides from newly synthesized MHCII and edits the peptide repertoire. Determinants of susceptibility of peptide/MHCII complexes to DM remain controversial, however. Here we have measured peptide dissociation in the presence and the absence of DM for 36 different complexes of varying intrinsic stability. We found large variations in DM susceptibility for different complexes using either soluble or full-length HLA-DM. The DM effect was significantly less for unstable complexes than for stable ones, although this correlation was modest. Peptide sequence- and allele-dependent interactions along the entire length of the Ag binding groove influenced DM susceptibility. We also observed differences in DM susceptibility during peptide association. Thus, the peptide repertoire displayed to CD4(+) T cells is the result of a mechanistically complicated editing process and cannot be simply predicted from the intrinsic stability of the complexes in the absence of DM.

Relationship between kinetic stability and immunogenicity of HLA-DR4/peptide complexes.

Immunodominant T cell epitopes from the autoantigen human cartilage glycoprotein 39 have previously been mapped in the context of HLA-DR*0401 and *0402, using mice expressing HLA-DR4 transgenes. We measured the dissociation rates of these epitopes from soluble recombinant DR*0401 and DR*0402 to assess the relationship between peptide/HLA-DR4 kinetic stability and immunogenicity. Experiments were performed at endosomal pH (5.5) and at cell surface pH (7), in the absence and presence of soluble recombinant HLA-DM (sDM). All (4/4) immunodominant peptide/HLA-DR complexes exhibit dissociation half-times of 1h to several days. In contrast, most (3/4) non-immunodominant complexes dissociate with half-times <30 min under at least one of these conditions. Interestingly, a complex which is stable except in the presence of HLA-DM at pH 5.5 is immunogenic only following peptide immunization, while a complex which is stable at acidic but not at neutral pH, is non-immunogenic following either whole protein or peptide immunization. These data indicate that kinetic stability of peptide/MHC complexes in vivo is a key determinant of immunogenicity.