Full-length rat amylin forms fibrils following substitution of single residues from human amylin.

Pancreatic amyloid deposits, composed of the 37 amino acid residue peptide amylin, represent an integral part of type 2 diabetes mellitus pathology. Human amylin (hA) forms fibrils in vitro and is toxic to cultured pancreatic islet beta-cells. In contrast, rat amylin (rA) which differs from hA by only six amino acid residues in the central region of the peptide, residues 18-29, does not form fibrils and is not cytotoxic. To elucidate the role of individual residues in fibril formation, we have generated a series of full-length rA variants and examined their ability to form fibrils in vitro. Single-residue substitutions with amino acids from corresponding positions of the hA sequence, i.e. R18H, L23F, or V26I, were sufficient to render rA competent for fibril formation albeit at a small yield. Combining two or three of these substitutions generally increased the ability to produce fibrils. Variant rA fibril morphologies were examined by negative stain electron microscopy and found to be similar to those generated by hA itself. Bulk assays, i.e. involving thioflavin-T fluorescence and sedimentation, showed that the amount of fibril formation was relatively small for these rA variants when compared to hA under the same conditions. Fibril growth was demonstrated by time-lapse atomic force microscopy, and MALDI-TOF mass spectrometry was used to verify that fibrils consisted of full-length peptide. Our observations confirm previous reports that the three proline residues play a dominant negative role in fibril formation. However, their presence is not sufficient to completely abolish the ability of rA to form fibrils, as each of the other three implicated residues (i.e. R18, L23 and V26) also has a dominant modulating effect.

Characterization by Mass Spectroscopy of a 10 kDa c-554 Cytochrome from the Green Sulfur Bacterium Chlorobium tepidum.

Preparative isoelectric focusing was used to isolate a type c cytochrome from photosynthetic membranes of the green sulfur bacterium Chlorobium tepidum. The purified protein showed a molecular weight of 10 kDa according to SDS-PAGE and ESI mass spectrometry. The absorption spectrum in the visible range is typical of a cytochrome with peaks at 420, 525.2 and 554.4 nm. Cleavage by either trypsin or endoproteinase lys-C of the isolated cytochrome combined with tandem mass spectrometry and Edman sequencing yielded a sequence perfectly matching parts of the recently sequenced genome of C. tepidum.

Essential role for cholesterol in the delivery of exogenous antigens to the MHC class I-presentation pathway.

Cross-presentation, which is crucial for the generation of immunity against virus-infected and tumor cells, requires exogenous antigens to be internalized into antigen-presenting cells (APCs) followed by translocation to the cytosol by unknown mechanisms. One important entry route for such antigens is macropinocytosis. We here describe that cholesterol is essential for cross-presentation of antigens loaded via macropinocytosis into APCs. Modification of antigens by palmitoylation to target antigens to cholesterol-enriched plasma membrane domains resulted in a dramatically increased T cell activation. These results define cholesterol as an essential factor for cross-presentation and suggest that specific modification of antigens to increase their affinity for cholesterol may be utilized to enhance immunity.

TOR regulates late steps of ribosome maturation in the nucleoplasm via Nog1 in response to nutrients.

The protein kinase TOR (target of rapamycin) controls several steps of ribosome biogenesis, including gene expression of rRNA and ribosomal proteins, and processing of the 35S rRNA precursor, in the budding yeast Saccharomyces cerevisiae. Here we show that TOR also regulates late stages of ribosome maturation in the nucleoplasm via the nuclear GTP-binding protein Nog1. Nog1 formed a complex that included 60S ribosomal proteins and pre-ribosomal proteins Nop7 and Rlp24. The Nog1 complex shuttled between the nucleolus and the nucleoplasm for ribosome biogenesis, but it was tethered to the nucleolus by both nutrient depletion and TOR inactivation, causing cessation of the late stages of ribosome biogenesis. Furthermore, after this, Nog1 and Nop7 proteins were lost, leading to complete cessation of ribosome maturation. Thus, the Nog1 complex is a critical regulator of ribosome biogenesis mediated by TOR. This is the first description of a physiological regulation of nucleolus-to-nucleoplasm translocation of pre-ribosome complexes.

Plasmodium falciparum possesses a cell cycle-regulated short type replication protein A large subunit encoded by an unusual transcript.

DNA replication in Plasmodium parasites takes place at multiple distinct points during their complex life cycle in the mosquito and vertebrate hosts. Although several parasite proteins involved in DNA replication have been described, the various mechanisms engaged in DNA metabolism of this major pathogen remain largely unexplored. As a step toward understanding this complex network, we describe the identification of Plasmodium falciparum replication protein A large subunit (pfRPA1) through affinity purification and mass spectral analysis of a purified 55-kDa factor. Gel retardation experiments revealed that pfRPA is the major single-stranded DNA binding activity in parasite protein extracts. The activity was expressed in a cell cycle-dependent manner with peak activities in late trophozoites and schizonts, thus correlating with the beginning of chromosomal DNA replication. Accordingly, the pfrpa1 message was detected in parasites 20-24 h post-invasion which is in agreement with the expression of other P. falciparum DNA replication genes. Our results show that pfRPA1 is encoded by an unusual 6.5-kb transcript containing a single open reading frame of which only the C-terminal 42% of the deduced protein sequence shows homologies to other reported RPA1s. Like the orthologues of other protozoan parasites, pfRPA1 lacks the N-terminal protein interaction domain and is thus remarkably smaller than the RPA1s of higher eukaryotes.

Quantitation of changes in protein phosphorylation: a simple method based on stable isotope labeling and mass spectrometry.

Reversible protein phosphorylation plays an important role in many cellular processes. However, a simple and reliable method to measure changes in the extent of phosphorylation is lacking. Here, we present a method to quantitate the changes in phosphorylation occurring in a protein in response to a stimulus. The method consists of three steps: (i) enzymatic digestion in H(2)16O or isotopically enriched H(2)18O to label individual pools of differentially phosphorylated proteins; (ii) affinity selection of phosphopeptides from the combined digests by immobilized metal-affinity chromatography; and (iii) dephosphorylation with alkaline phosphatase to allow for quantitation of changes of phosphorylation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We applied this strategy to the analysis of the yeast nitrogen permease reactivator protein kinase involved in the target of rapamycin signaling pathway. Alteration in the extent of phosphorylation at Ser-353 and Ser-357 could be easily assessed and quantitated both in wild-type yeast cells treated with rapamycin and in cells lacking the SIT4 phosphatase responsible for dephosphorylating nitrogen permease reactivator protein. The method described here is simple and allows quantitation of relative changes in the level of phosphorylation in signaling proteins, thus yielding information critical for understanding the regulation of complex protein phosphorylation cascades.

Characterization of the matrilin coiled-coil domains reveals seven novel isoforms.

Matrilins constitute a family of four oligomeric extracellular proteins that are involved in the development and homeostasis of cartilage and bone. To reveal their homo- and heterotypic oligomerization propensities, we analyzed the four human matrilin coiled-coil domains by biochemical and biophysical methods. These studies not only confirmed the homo- and heterotypic oligomerization states reported for the full-length proteins but revealed seven novel matrilin isoforms. Specific heterotrimeric interactions of variable chain stoichiometries were observed between matrilin-1 and matrilin-2, matrilin-1 and matrilin-4, and matrilin-2 and matrilin-4. In addition, matrilin-1 formed two different specific heterotetramers with matrilin-3. Interestingly, a distinct heterotrimer consisting of three different chains was formed between matrilin-1, matrilin-2, and matrilin-4. No interactions, however, were observed between matrilin-2 and matrilin-3 or between matrilin-3 and matrilin-4. Both homo- and heterotypic oligomers folded into parallel disulfide-linked structures, although coiled-coil formation was not dependent on disulfide bridge formation. Our results indicate that the heterotypic preferences seen for the matrilin coiled-coil domains are the result of the packing of the hydrophobic core rather than ionic interactions. Mass spectrometry revealed that the concentrations of the individual chains statistically determined the stoichiometry of the heteromers, suggesting that formation of the different matrillin chain combinations is controlled by expression levels.

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of the bacterial pathogen Bartonella henselae.

Bartonella henselae is an emerging zoonotic pathogen causing a wide range of disease manifestations in humans. In this study, we report on the analysis of the sarcosine-insoluble outer membrane fraction of B. henselae ATCC 49882 Houston-1 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) and two-dimensional nonequilibrium pH gradient polyacrylamide gel electrophoresis (2-D NEPHGE). Protein species were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and subsequent database query against the B. henselae genome sequence. Subcellular fractionation, application of the ionic detergent lauryl sarcosine, assessment of trypsin sensitivity, and heat modifiability of surface-exposed proteins represented valuable tools for the analysis of the outer membrane subproteome of B. henselae. 2-D NEPHGE was applied to display and catalogue a substantial number of proteins associated with the B. henselae sarcosine-insoluble outer membrane fraction, resulting in the establishment of a first 2-D reference map of this compartment. Thus, 53 distinct protein species associated with the outer membrane subproteome fraction were identified. This study provides novel insights into the membrane biology and the associated putative virulence factors of this pathogen of increasing medical importance.