The allograft inflammatory factor-1 in Creutzfeldt-Jakob disease brains.

The allograft inflammatory factor-1 (AIF-1) is a 17-kDa IFN-gamma inducible Ca(2+)-binding EF-hand protein that is encoded within the HLA class III genomic region and is involved in immune dysfunction and smooth muscle cell activation. We used immunohistochemistry double labelling experiments to analyse the spatial distribution and cell-type-specific localization of AIF-1 in the brains of patients who died as a result of sporadic Creutzfeldt-Jakob disease (CJD) and neuropathologically unaltered controls. Significantly more AIF-1 immunoreactive macrophages/microglial cells and, interestingly, neurones were observed in CJD patients compared to controls. Western blotting confirmed more prominent AIF-1 immunoreactive bands of approximately 50 kDa in four CJD patients compared to three controls. Chaotropic SDS-PAGE of the recombinant AIF-1 resulted in almost complete reduction of the 50 kDa band and mass spectrometry revealed only AIF-1-specific tryptic protein fragments suggesting that trimerized AIF-1 is the predominant form in vivo. Finally, we analysed mechanisms of neuronal AIF-1 induction. Following H2O2 challenge, a model of general cell stress, we observed the gradual induction of AIF-1 and, more interestingly, release to the supernatant of SKNSH neurones. Parallel reverse transcriptase polymerase chain reaction and sequencing was used to confirm AIF-1 mRNA expression.

Combining computer algorithms with experimental approaches permits the rapid and accurate identification of T cell epitopes from defined antigens.

The identification of T cell epitopes from immunologically relevant antigens remains a critical step in the development of vaccines and methods for monitoring of T cell responses. This review presents an overview of strategies that employ computer algorithms for the selection of candidate peptides from defined proteins and subsequent verification of their in vivo relevance by experimental approaches. Several computer algorithms are currently being used for epitope prediction of various major histocompatibility complex (MHC) class I and II molecules, based either on the analysis of natural MHC ligands or on the binding properties of synthetic peptides. Moreover, the analysis of proteasomal digests of peptides and whole proteins has led to the development of algorithms for the prediction of proteasomal cleavages. In order to verify the generation of the predicted peptides during antigen processing in vivo as well as their immunogenic potential, several experimental approaches have been pursued in the recent past. Mass spectrometry-based bioanalytical approaches have been used specifically to detect predicted peptides among isolated natural ligands. Other strategies employ various methods for the stimulation of primary T cell responses against the predicted peptides and subsequent testing of the recognition pattern towards target cells that express the antigen.

A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex class I antigenic peptides.

Peptides presented to cytotoxic T lymphocytes by the class I major histocompatability complex are 8-11 residues long. Although proteasomal activity generates the precise C termini of antigenic epitopes, the mechanism(s) involved in generation of the precise N termini is largely unknown. To investigate the mechanism of N-terminal peptide processing, we used a cell-free system in which two recombinant ornithine decarboxylase (ODC) constructs, one expressing the native H2-K(b)-restricted ovalbumin (ova)-derived epitope SIINFEKL (ODC-ova) and the other expressing the extended epitope LESIINFEKL (ODC-LEova), were targeted to degradation by 26 S proteasomes followed by import into microsomes. We found that the cleavage specificity of the 26 S proteasome was influenced by the N-terminal flanking amino acids leading to significantly different yields of the final epitope SIINFEKL. Following incubation in the presence of purified 26 S proteasome, ODC-LEova generated largely ESIINFEKL that was efficiently converted to the final epitope SIINFEKL following translocation into microsomes. The conversion of ESIINFEKL to SIINFEKL was strictly dependent on the presence of H2-K(b) and was completely inhibited by the metalloaminopeptidase inhibitor 1,10-phenanthroline. Importantly, the converting activity was resistant to a stringent salt/EDTA wash of the microsomes and was only apparent when transport of TAP, the transporter associated with antigen processing, was facilitated. These results strongly suggest a crucial role for a luminal endoplasmic reticulum-resident metalloaminopeptidase in the N-terminal trimming of major histocompatability complex class I-associated peptides.

Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels. selective targeting of endothelial regulatory protein pigpen.

Tumor microvessels differ in structure and metabolic function from normal vasculature, and neoangiogenesis is associated with quantitative and qualitative changes in expression of endothelial proteins. Such molecules could serve as molecular addresses differentiating the tumor vasculature from those of the normal brain. We have applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against transformed endothelial cells as a complex target to select single-stranded DNA-ligands (aptamers) that function as histological markers to detect microvessels of rat experimental glioma, a fatal brain tumor that is highly vascularized. Both the SELEX selection procedure as well as subsequent deconvolution-SELEX were analyzed by fluorescence based methods (flow cytometry and fluorescence microscopy). Of 25 aptamers analyzed, one aptamer was selected that selectively bound microvessels of rat brain glioblastoma but not the vasculature of the normal rat brain including peritumoral areas. The molecular target protein of aptamer III.1 was isolated from endothelial cells by ligand-mediated magnetic DNA affinity purification. This protein was identified by mass spectrometry as rat homologue of mouse pigpen, a not widely known endothelial protein the expression of which parallels the transition from quiescent to angiogenic phenotypes in vitro. Because neoangiogenesis, the formation of new blood vessels, is a key feature of tumor development, the presented aptamer can be used as a probe to analyze pathological angiogenesis of glioblastoma. The presented data show that pigpen is highly expressed in tumor microvessels of experimental rat brain glioblastoma and may play an important role in warranting blood supply, thus growth of brain tumors.

Broadband detection electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to reveal enzymatically and chemically induced deamidation reactions within peptides.

Among the numerous forms of chemical degradation of peptides or proteins, deamidation is one of the alterations observed most frequently. In this irreversible reaction, a glutamine or an asparagine side chain is hydrolyzed to glutamic acid or aspartic acid, respectively (conversion of NH2 to OH). Besides its influence in the deterioration of biotechnological and food products, deamidation represents a defined posttranslational modification reaction with respect to proteomics. Here mass spectrometric techniques play a leading role in determining posttranslational modifications. However, not all mass spectrometers are able to resolve signal differences of 0.0193 Da (mass difference of 12CO vs 13CNH) for singly charged molecules, the mass difference between the first isotopic signal of an asparagine/glutamine-containing peptide and the monoisotopic signal of the corresponding partially deamidated aspartate/glutamate derivative. To detect partial deamidation within peptides, advantage has been taken of the ability of Fourier transform ion cyclotron resonance mass spectrometry to perform very high mass resolution. In this work, we investigated up to triply charged ions produced by electrospray ionization using direct infusion. Although the special heterodyne detection mode enables higher mass resolution than the routinely used broadband detection, often only a small mass window can be investigated. Using broadband detection, we were able to resolve ions with a difference of m/z 0.0064 to detect partially deamidated peptides formed either enzymatically or under acidic and basic conditions.