Long-term survival but impaired homeostatic proliferation of Naïve T cells in the absence of p56lck.

Interactions between the T cell receptor (TCR) and major histocompatibility complex antigens are essential for the survival and homeostasis of peripheral T lymphocytes. However, little is known about the TCR signaling events that result from these interactions. The peripheral T cell pool of p56lck (lck)-deficient mice was reconstituted by the expression of an inducible lck transgene. Continued survival of peripheral naïve T cells was observed for long periods after switching off the transgene. Adoptive transfer of T cells from these mice into T lymphopoienic hosts confirmed that T cell survival was independent of lck but revealed its essential role in TCR-driven homeostatic proliferation of naïve T cells in response to the T cell-deficient host environment. These data suggest that survival and homeostatic expansion depend on different signals.

Differential overexpression of SERPINA3 in human prion diseases.

Prion diseases are fatal neurodegenerative disorders with sporadic, genetic or acquired etiologies. The molecular alterations leading to the onset and the spreading of these diseases are still unknown. In a previous work we identified a five-gene signature able to distinguish intracranially BSE-infected macaques from healthy ones, with SERPINA3 showing the most prominent dysregulation. We analyzed 128 suitable frontal cortex samples, from prion-affected patients (variant Creutzfeldt-Jakob disease (vCJD) n = 20, iatrogenic CJD (iCJD) n = 11, sporadic CJD (sCJD) n = 23, familial CJD (gCJD) n = 17, fatal familial insomnia (FFI) n = 9, Gerstmann-Sträussler-Scheinker syndrome (GSS)) n = 4), patients with Alzheimer disease (AD, n = 14) and age-matched controls (n = 30). Real Time-quantitative PCR was performed for SERPINA3 transcript, and ACTB, RPL19, GAPDH and B2M were used as reference genes. We report SERPINA3 to be strongly up-regulated in the brain of all human prion diseases, with only a mild up-regulation in AD. We show that this striking up-regulation, both at the mRNA and at the protein level, is present in all types of human prion diseases analyzed, although to a different extent for each specific disorder. Our data suggest that SERPINA3 may be involved in the pathogenesis and the progression of prion diseases, representing a valid tool for distinguishing different forms of these disorders in humans.

Identification and cloning of human chaperonin 10 homologue.

We have identified a heat-shock-inducible 10 kDa protein in the human hepatoma cell line HepG2. The total RNA extracted from the heat-shocked cells was amplified by reverse transcription PCR (polymerase chain reaction) using 21 5' and 18 3' oligonucleotides of rat cpn10 (chaperonin10) cDNA as primers. Sequencing of the above PCR fragment showed a very high homology between human, bovine and rat cpn10 cDNA. The predicted amino acid sequence revealed a 100% identity with the bovine homologue.

Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.

Mycobacterial Cpn10 promotes recognition of the mammalian homologue by a mycobacterium-specific antiserum.

Self-tolerance, a key feature of the immune system, is still a matter of intense debate. We give here evidence for a peculiar behavior of an antiserum against Mycobacterium tuberculosis chaperonin 10 (m-Cpn10), which could have implications for the mechanism of self-recognition by antibodies against non-self. We show that this antiserum can interact in terms of both inhibition of biological activity and physical association (immunoprecipitation), with the mammalian homologue of m-Cpn10, but only if the bacterial protein is present. Several lines of evidence led us to exclude that the two proteins physically associate to form heterocomplexes: (1) the behavior of the antiserum was not shared by a monoclonal antibody against m-Cpn10; (2) a matrix selective for human Cpn10 (h-Cpn10) did not co-purify m-Cpn10; (3) the distribution pattern in non-denaturing isoelectric focusing of labeled m-Cpn10 was not altered by the presence of the unlabeled h-Cpn10. We conclude therefore that the antiserum against M. tuberculosis Cpn10 also recognizes mammalian Cpn10, with an affinity/avidity regulated by the mycobacterial protein, or by the promotion of hetero-oligomerization. This emergence of self-recognition in the presence of M. tuberculosis Cpn10 could imply a breaking of self-tolerance in situations of infection or vaccination.

Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein.

To identify molecular interaction partners of the cellular prion protein (PrP(C)), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP(C). Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP(C) in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrP(Sc) replication. Our findings raise the possibility that N-CAM may join with PrP(C) in carrying out some as yet unidentified physiologic cellular function.

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3' end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A2660, which lies in a sequence that is highly conserved in all species.

Expression in Escherichia coli, purification and functional activity of recombinant human chaperonin 10.

We have recently reported the cloning of a cDNA coding for a stress inducible human chaperonin 10. The protein was shown to possess 100% identity with the bovine homologue and a single amino acid replacement (glycine to serine at position 52) compared to rat chaperonin 10. Here we report the heterologous expression of human chaperonin 10 in Escherichia coli, its purification and its functional characterization. The recombinant protein was purified to homogeneity as judged by different analytical techniques, and mass spectrometry analysis showed a MW of 10,801 Da in agreement with the predicted sequence. This molecular weight accounts for a protein which is not modified post-translationally. In fact, natural rat chaperonin 10 has been shown to be acetylated at the N-terminus, a feature suggested to be important for targeting and functional activity. Here we show that recombinant human chaperonin 10 is fully active in assisting the chaperonin 60 GroEL in the refolding of denatured yeast enolase, thereby showing that, at least in the present system, post-translational acetylation is not necessary for its activity.

Anti-GM1 IgM antibodies in motor neuron disease and neuropathy.

We found anti-GM1 IgM antibodies in 23% of 56 patients with motor neuron disease (MND), in 19% of 69 patients with neuropathy, and in 7% of 107 controls with other neurologic and nonneurologic diseases. Most of these patients had anti-GM1 IgM antibody titers of 1:80 or less; slightly higher antibody titers (up to 1:640) were found in 3 patients, 1 with MND and 2 with neuropathy, and very high titers (1:20,480) in a patient with MND and an IgM kappa M protein that reacted with GM1, GD1b, and asialo GM1. Six other patients with anti-GM1 IgM that also bound to GD1b. Reactivity with GD1b did not correlate with anti-GM1 titers but was only present in patients with MND or neuropathy. Anti-GM1 IgM antibodies may be a normal constituent of the human antibody repertoire but their frequency and, in some cases, their levels are higher in patients with MND and neuropathy. The origin and the pathogenetic role of these antibodies in neural impairment remain to be established.

Microglia convert aggregated amyloid-ß into neurotoxic forms through the shedding of microvesicles.

Alzheimer's disease (AD) is characterized by extracellular amyloid-ß (Aß) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aß species, rather than insoluble fibrils, are the most toxic forms of Aß. Preventing soluble Aß formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aß species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aß forms trafficked to MVs after Aß internalization into microglia. MV neurotoxicity was neutralized by the Aß-interacting protein PrP and anti-Aß antibodies, which prevented binding to neurons of neurotoxic soluble Aß species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.

Substrate recognition by ribosome-inactivating protein studied by molecular modeling and molecular electrostatic potentials.

A computer model of dianthin 30, a type 1 ribosome-inactivating protein (RIP), is constructed by homology modeling using two known X-ray structures; a type 1 RIP, pokeweed antiviral protein (PAP), and chain A of a type 2 RIP, ricin. The 3D structure is refined by molecular dynamics and its binding site compared with those of PAP and ricin using molecular electrostatic potential mapping. The differences in the maps obtained clearly show how, despite the similarity of the topology of the binding site, differences in electrostatic potential can account for the experimentally observed differences in substrate recognition and binding. This demonstrates the potential of these techniques for guiding further experimental analyses.

Heterologous expression, purification, activity and conformational studies of different forms of dianthin 30.

Dianthin 30, a ribosome inactivating protein (RIP) from Dianthus caryophyllus, has been expressed in Escherichia coli. Heterologous expression of a deletion mutant dianthin 30 delta 255-270 resulted in the production of a protein identical to carnation mature dianthin 30, including the absence at the carboxy-terminal of a putative 16 amino acid long pro-signal peptide. The production of a form of dianthin 30, which includes the pro-signal, is described as well. Both dianthin 30 delta 255-270 and dianthin 30 expressed in E. coli are mainly localized (90%) in the soluble fraction. Dianthin 30 delta 255-270 and dianthin 30 have been purified to homogeneity and were shown to inhibit protein synthesis in vitro with an IC50 of 8 and of 11 ng/ml, respectively. Secondary structure analysis, carried out by circular dichroism spectroscopy, indicated that the naturally occurring and the recombinant forms of dianthin 30 and dianthin 30 delta 255-270 possess the same secondary structure composition, accounting for an alpha + beta type architecture. RIPs as immunotoxins in clinical trial and as mitotoxins in experimental models have been extremely efficacious. In addition, growing evidence indicates their effective use as antiviral agents, including in HIV-1 infection. These data indicate the ability to produce either chemically linked or recombinant fusion proteins with dianthin 30 and cell-binding ligands for production of new reagents for clinical and experimental use.

Expression and activity of pre-dianthin 30 and dianthin 30.

Dianthin 30 is a ribosome inactivating protein (RIP 1) found in different tissues of the carnation (Dianthus caryophyllus). Recently we have isolated and sequenced a cDNA clone from a lambda gt11 expression library [Legname et al. (1991) Biochim. Biophys. Acta 1090, 119-122]. Here we describe specific PCR amplifications of either the full length pre-dianthin 30 or dianthin 30, the mature polypeptide lacking the 23 amino acid signal peptide. In vitro expression of both proteins in reticulocyte lysate generated products of the expected molecular weight. Moreover, the activity of both proteins has been evaluated confirming the characteristics of the natural product. A first attempt to produce recombinant dianthin 30 in Escherichia coli is described.

Folding of prion protein to its native alpha-helical conformation is under kinetic control.

The recombinant mouse prion protein (MoPrP) can be folded either to a monomeric alpha-helical or oligomeric beta-sheet-rich isoform. By using circular dichroism spectroscopy and size-exclusion chromatography, we show that the beta-rich isoform of MoPrP is thermodynamically more stable than the native alpha-helical isoform. The conformational transition from the alpha-helical to beta-rich isoform is separated by a large energetic barrier that is associated with unfolding and with a higher order kinetic process related to oligomerization. Under partially denaturing acidic conditions, MoPrP avoids the kinetic trap posed by the alpha-helical isoform and folds directly to the thermodynamically more stable beta-rich isoform. Our data demonstrate that the folding of the prion protein to its native alpha-helical monomeric conformation is under kinetic control.

Evidence for GroES acting as a transcriptional regulator.

Cochaperonins (cpn10) assist chaperonins (cpn60) in promoting folding and assembly of other proteins. Upon expression of Mycobacterium tuberculosis cpn10 in Escherichia coli we have purified a polypeptide which, through amino acid sequencing, was identified as the endogenous E. coli 10K-S protein. Subsequent studies showed that its expression was specifically upregulated upon cloning of different members of the cpn10 family, including GroES, the E. coli cpn10. Pulse-chase experiments demonstrated that 10K-S is but one of several proteins whose expression is modulated upon cloning of cpn10. Up-regulation of 10K-S was also observed after exposure of normal cells, but not of groES- mutants, to elevated temperatures (42 degrees C). This allowed us to define 10K-S as a heat-shock protein (hsp) whose expression is dependent on the production of another hsp, GroES. Northern blot experiments showed that enhanced expression of 10K-S was consequent to increased accumulation of transcripts and that groES- mutants were devoid even of baseline levels of transcripts both at 37 degrees C and after temperature upshift. These results show that GroES, in addition to its established role in assisting protein folding may act as a transcriptional regulator and is likely to play an important role in modulating gene expression particularly in those conditions, like the stress response, in which its production is greatly enhanced.

Anti-CD30 immunotoxins with native and recombinant dianthin 30.

Immunotoxins were prepared with a Ber-H2 (anti-CD30) monoclonal antibody and native or recombinant dianthin 30, a ribosome-inactivating protein from Dianthus caryophyllus (carnation). Both immunotoxins selectively inhibited protein synthesis by CD30+ cell lines D430B (lymphoblastoid, infected with Epstein-Barr virus), L428 and L540 (both from Hodgkin's lymphoma). IC50 values (concentrations, as dianthin, causing 50% inhibition) ranged from 324 pM to 479 pM (immunotoxin with native dianthin 30) or from 45 pM to 182 pM (immunotoxin with recombinant dianthin 30). The effect of either immunotoxin on protein synthesis by the CD30+ cell line K562 (from a chronic myeloid leukaemia) was not different from that of free dianthin (IC50 higher than nM).

Cochaperonins are histone-binding proteins.

Cochaperonins (cpn10) assist chaperonins (cpn60) in mediating folding of polypeptide substrates in an ATP-dependent reaction. Moreover, they have been shown to be secretory products of living cells and to perform discrete biological activities without the need to interact with cpn60. Here, we have investigated the possible existence of cellular cpn10 binding sites that could mediate such activities. For this purpose, we performed binding studies with iodinated cpn10 on whole cells and on electrophoretically separated eukaryotic cell lysates. The former studies yielded negative results, whereas in the latter binding to several proteins was detected. These proteins were identified as being histones. Binding was observed to all core histones (H2A, H2B, H3 and H4) and, although weaker, to the linker histone H1 as well. These results show that cpn10 are histone-binding proteins.

Copper-catalyzed oxidation of the recombinant SHa(29-231) prion protein.

Metal-catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidized protein molecules. PrP contains a histidine-rich octarepeat domain that binds copper. Because copper-binding histidine residues are particularly prone to metal-catalyzed oxidation, we investigated the effect of this reaction on the recombinant prion protein SHaPrP(29-231). Using Cu2+/ascorbate, we oxidized SHaPrP(29-231) in vitro. Oxidation was demonstrated by liquid chromatography/mass spectrometry, which showed the appearance of protein species of higher mass, including increases in multiples of 16, characteristic of oxygen incorporation. Digestion studies using Lys C indicate that the 29-101 region, which includes the histidine-containing octarepeats, is particularly affected by oxidation. Oxidation was time- and copper concentration-dependent and was evident with copper concentrations as low as 1 microM. Concomitant with oxidation, SHaPrP(29-231) suffered aggregation and precipitation, which was nearly complete after 15 min, when the prion protein was incubated at 37 degrees C with a 6-fold molar excess of Cu2+. These findings indicate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structural transition leading to aggregation.

Motor neuron disease in a patient with a monoclonal IgMk directed against GM1, GD1b, and high-molecular-weight neural-specific glycoproteins.

In a patient with motor neuron disease and benign IgMk monoclonal gammopathy, the M protein reacted with the glycolipids GM1, GD1b, and asialo GM1 and, by immunoblot, with some high-molecular-weight neural-specific glycoproteins. The main reactive bands had an approximate molecular weight of 250 and 400 kd, were most concentrated in the spinal cord, and were also bound by the lectin peanut agglutinin. The presence of the Ga1(beta 1-3)Ga1NAc epitope on these neural-specific glycoproteins may help to explain the selective neurological impairment of the patient.

Genetic grafting of membrane-acting peptides to the cytotoxin dianthin augments its ability to de-stabilize lipid bilayers and enhances its cytotoxic potential as the component of transferrin-toxin conjugates.

Three chimeric proteins were obtained by fusing together the dianthin gene and DNA fragments encoding for the following membrane-acting peptides: the N-terminus of protein G of the vesicular stomatitis virus (KFT25), the N terminus of the HA2 hemagglutinin of influenza virus (pHA2), and a membrane-acting peptide (pJVE). Chimeric dianthins (KFT25DIA, pHA2DIA and pJVEDIA) retained full enzymatic activity in cell-free assays and showed increased ability to induce pH-dependent calcein release from large unilamellar vesicles (LUVs). pHA2DIA and pJVEDIA also showed faster kinetics of interaction with LUVs, while KFT25DIA and pHA2DIA displayed a reduced cytotoxicity as compared to wild-type dianthin. Conjugates made by chemically cross-linking KFT25DIA or pJVEDIA and human transferrin (Tfn) showed greater cell-killing efficiency than conjugates of Tfn and wild-type dianthin. As a consequence, by fusion of membrane-acting peptides to the dianthin sequence the specificity factor (i.e., the ratio between non-specific and specific toxicity) of Tfn-KFT25DIA, Tfn-pHA2DIA and Tfn-pJVEDIA was increased with respect to that of Tfn-based conjugates made with wild-type dianthin. Taken together, our results suggest that genetic fusion of membrane-acting peptides to enzymatic cytotoxins results in the acquisition of new physico-chemical properties exploitable for designing new recombinant cytotoxins and to tackle cell-intoxication mechanisms.