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The present study was undertaken to identify proteins interacting with PrP(C) that could provide new insights into its physiological functions and pathological role. Human PrP(C) was expressed in prion protein-deficient murine hippocampus (HpL3-4) neuronal cells. The PrP(C) along with its interacting proteins were affinity purified using STrEP-Tactin-chromatography, in-gel digested, and identified by Q-TOF MS/MS analysis. Forty-three proteins appeared to interact with PrP(C) in this neuronal cell line. Of these, 15 were already known for their interaction with PrP(C) or PrP(Sc), while 28 new proteins were identified. Interaction of a novel interacting partner of GTPase family-Rab7a, having a suggested role in vesicle trafficking, was further investigated using confocal laser scanning microscopy and reverse coimmunoprecipitation. Both reverse coimmunoprecipitation and immunofluorescence results confirmed potential interaction of Rab7a with the PrP(C). siRNA against the Rab7a gene decreased expression of Rab7a protein, in PrP(C) expressing HpL3-4 and SH-SY5Y cells. This depleted Rab7a expression led to the enhanced accumulation of PrP(C) in Rab9 positive endosomal compartments and consequently an increased colocalization between PrP(C)/Rab9. However, the Rab9 accumulated PrP(C) remained sensitive to proteinase-K digestion. The work described demonstrated for the first time that Rab7a interacts with PrP(C) and highlighted the involvement of endosomal compartments in the trafficking and regulation of PrP(C).

Multipotent adult germline stem cells (maGSCs) are pluripotent cells that can be differentiated into somatic cells of the three primary germ layers. To highlight the protein profile changes associated with stem cell differentiation, retinoic acid (RA) treated mouse stem cells (maGSCs and ESCs) were compared to nontreated stem cells. 2-DE and DIGE reference maps were created, and differentially expressed proteins were further processed for identification. In both stem cell types, the RA induced differentiation resulted in an alteration of 36 proteins of which 18 were down-regulated and might be potential pluripotency associated proteins, whereas the other 18 proteins were up-regulated. These might be correlated to stem cell differentiation. Surprisingly, eukaryotic initiation factor 5A (Eif5a), a protein which is essential for cell proliferation and differentiation, was significantly down-regulated under RA treatment. A time-dependent investigation of Eif5a showed that the RA treatment of stem cells resulted in a significant up-regulation of the Eif5a in the first 48 h followed by a progressive down-regulation thereafter. This effect could be blocked by the hypusination inhibitor ciclopirox olamine (CPX). The alteration of Eif5a hypusination, as confirmed by mass spectrometry, exerts an antiproliferative effect on ESCs and maGSCs in vitro, but does not affect the cell pluripotency. Our data highlights the important role of Eif5a and its hypusination for stem cell differentiation and proliferation.

Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which can reflect a physiological and pathological state of the central nervous system. In our present study, we show CSF protein patterns from patients with the two most frequent subtypes of sporadic Creutzfeldt-Jakob disease (sCJD) defined by the codon 129 genotype (MM, MV, and VV) and the protease-resistant form of prion protein (type 1 and type 2). The densitometric analysis of 2D gels showed up-regulation of 27 and down-regulation of 3 proteins in the MM-sCJD as well as the up-regulation of 24 proteins in the VV-sCJD as compared to nondemented control. Almost 40% of sCJD specific regulated proteins in CSF are involved in glucose metabolism, regardless of the codon 129 polymorphism. The increase in CSF levels of lactate dehydrogenase (LDH), glucose-6-phosphate isomerase (G6PI), and fructose-bisphosphate aldolase A (ALDOA) were validated on a larger group of sCJD patients including three possible codon 129 polymorphism carriers and three control groups consisting of nondemented, neurological cases as well as patients suffering from Alzheimer's disease or vascular dementia. Subsequently, the abundance of these glycolytic enzymes in the brain as well as their cellular localization were determined. This study demonstrates for the first time the implication of G6PI in prion-induced pathology as well as its cellular translocalization in sCJD. The identification of sCJD-regulated proteins in CSF of living symptomatic patients in our study can broaden our knowledge about pathological processes occurring in sCJD, as they are still not fully understood.

Aspergillus fumigatus is the common cause of allergic broncho-pulmonary aspergillosis (ABPA) and most of the allergens have been described from its secreted fraction. In the present investigation, germinating conidial cytosolic proteins of A. fumigatus were extracted from a 16 h culture. The proteome from this fraction was developed, and immuno-blots were generated using pooled ABPA patients' sera. Well separated Immunoglobulin-E (IgE) and Immunoglobulin-G (IgG) reactive spots were picked from corresponding 2DE gels and subjected to mass spectrometric analysis. As a result, 66 immuno-reactive proteins were identified from two geographically different strains (190/96 and DAYA) of A. fumigatus. Only 3 out of 66 proteins reacted with IgG, and the remaining 63 proteins were found to be IgE reactive. These 63 IgE-reactive cytosolic proteins from germinating conidia included 2 already known (Asp f12 and Asp f22) and 4 predicted allergens (Hsp88, Hsp70, malate dehydrogenase, and alcohol dehydrogenase) based on their homology with other known fungal allergens. In view of this, the panel of presently identified IgE-reactive novel proteins holds the potential of providing a basis for the wider diagnostic application in assay for allergic aspergillosis. We could demonstrate that recombinantly expressed proteins from this panel showed consistent reactivity with IgE of individual sera of ABPA patients. The recombinantly expressed proteins may also be useful in desensitization therapy of allergic disorders including ABPA.

The secreted proteomes of a three week old culture of an Indian (190/96) and a German (DAYA) Aspergillus fumigatus isolate were investigated for reactivity with IgG and/or IgE antibodies derived from pooled allergic broncho-pulmonary aspergillosis (ABPA) patients' sera. Two dimensional Western blotting followed by mass spectrometric analysis of the reactive protein spots revealed 35 proteins from the two A. fumigatus strains. There were seven known A. fumigatus allergens among them (Asp f1-4, Asp f9, Asp f10, and Asp f13/15), whereas three proteins displaying significant sequence similarity to known fungal allergens have been assigned as predicted allergens (Dipeptidyl-peptidase-V precursor, Nuclear transport factor 2, and Malate dehydrogenase, NAD-dependent). Eight IgG and IgE reactive proteins were common in both strains; however, 12 proteins specifically reacted in 190/96 and 15 in DAYA. Further testing with sera of 5 individual ABPA patients demonstrated that 12 out of 20 immunoreactive proteins of 190/96 strain of A. fumigatus had consistent reactivity with IgE. Seven of these proteins reacted with IgG also. The 25 of 35 identified proteins are novel with respect to immuno-reactivity with ABPA patients' sera and could form a panel of molecules to improve the currently existing less-sensitive diagnostic methods. Through expressing recombinantly, these proteins may also serve as a tool in desensibilization strategies.

Aspergillus fumigatus is a mold causing most of the invasive fungal lung infections in the immunocompromised host. In addition, the species is the causative agent of certain allergic diseases. Both in invasive and in allergic diseases, the conidial surface mediates the first contact with the human immune system. Thus, conidial surface proteins may be reasonable vaccine candidates as well as important allergens. To broaden the list of those antigens, intact viable Aspergillus conidia were extracted with mild alkaline buffer at pH 8.5 in the presence of a 1,3-beta-glucanase. The proteome of this fraction was separated by two- dimensional gel electrophoresis (2-DE) and analyzed by liquid chromatography coupled with tandem mass spectrometry. Altogether 26 different A. fumigatus proteins were identified, twelve of which contain a signal for secretion. Among these were the known major conidial surface protein rodlet A, one acid protease PEP2, one lipase, a putative disulfide isomerase and a putative fructose-1,6-biphosphatase. The known allergen Aspf 3 was identified among the proteins without a signal for secretion. On the basis of the recently annotated A. fumigatus genome (Nature 2005, 438, 1151-1156), proteome analysis is now a powerful tool to confirm expression of hypothetical proteins and, thereby to identify additional vaccine candidates and possible new allergens of this important fungal pathogen.

We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein-protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility.

The physiological role of the cellular prion protein (PrP (c)) is still not fully understood. Current evidence strongly suggests that PrP (c) overexpression in different cell lines sensitizes cells to apoptotic stimuli through a p53 dependent pathway. On the other hand, an expression of PrP (c) in PrP (c)-deficient cells undergoing apoptosis exhibited repeatedly antiapoptotic effects. Therefore, the presence/absence and/or the level of PrP (c) expression seem to be critical for the fluctuation between PrP (c)'s pro- and antiapoptotic properties. The present study examined whether an overexpression of PrP (c) itself, without addition of any apoptotic agent, can lead to proteome changes that might account for the higher responsiveness to apoptotic stimuli. Beyond this, we examined whether the sole introduction of PrP (c) into PrP (c)-deficient cells could be sufficient to up-regulate antiapoptotic proteins capable of mitigating apoptosis. For this purpose, we used two cell lines, one expressing [human embryonic kidney (HEK) 293 cells] and the other lacking (mouse neuronal PrP (c)-deficient cells) endogenous PrP (c). Protein profiling following transient PrP (c) overexpression in HEK 293 cells revealed a major PrP (c) involvement in regulation of proteins participating in energy metabolism and cellular homeostasis, whereas transient introduction of PrP (c) into mouse neuronal PrP (c)-deficient cells resulted mainly in the regulation of proteins involved in protection against oxidative stress and apoptosis. In addition, we report for the first time that PrP (c) overexpression influenced the regulation of several proteins known to have contributory roles in the pathogenesis of Alzheimer disease (AD). Revealing the correlation between presence/absence and/or different levels of PrP (c) expression with the regulation of certain cellular proteins might further contribute to our understanding of the complex role of PrP (c) in cell physiology.