Methylcrotonyl-CoA and geranyl-CoA carboxylases are involved in leucine/isovalerate utilization (Liu) and acyclic terpene utilization (Atu), and are encoded by liuB/liuD and atuC/atuF, in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to grow on acyclic monoterpenes (citronellol, citronellate, geraniol and geranylate), and on other methyl-branched compounds such as leucine or isovalerate. The catabolic pathway of citronellol (Atu, acyclic terpene utilization) enters that of leucine/isovalerate (Liu, leucine and isovalerate utilization) at the level of methylcrotonyl-CoA. Key enzymes of the combined pathways are geranyl-CoA carboxylase (GCase) and methylcrotonyl-CoA carboxylase (MCase).

Differential detection of S100A8 in transitional cell carcinoma of the bladder by pair wise tissue proteomic and immunohistochemical analysis.

The search for novel molecular markers of tumor invasion is vital if strategies are to become more effective in the diagnostic and prognostic management of transitional cell carcinoma of the bladder. Up to 50% of tumors detected at stage 1 (pT1) progress to a higher grade even after endoscopic surgical resection, and there are currently no protein markers of this aggressive, invasive phenotype. We have combined SELDI-TOF-MS, ClinProt magnetic bead enrichment, Nano-LC-ESI-ion trap tandem mass spectrometry and immunohistochemical analysis to the study of 12 invasive bladder cancer tissue biopsies paired with normal bladder tissue samples obtained from the same patients for the definition and identification of proteins up-regulated in the tumors.

Functional organization of the yeast proteome by systematic analysis of protein complexes.

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae.

AMP deaminase in rat brain: localization in neurons and ependymal cells.

The purine nucleotide cycle enzyme AMP deaminase (AMPD) catalyzes the irreversible hydrolytic deamination of AMP. The physiological function of the purine nucleotide cycle in the brain is unknown. In situ hybridization and immunocytochemical studies were performed to identify the regional and cellular expression of AMPD in rat brain with the goal of elucidating the neural function of the purine nucleotide cycle.

Identification of unusual amino acids in peptides using automated sequential Edman degradation coupled to direct detection by electrospray-ionization mass spectrometry.

The determination of the primary structure of peptides and proteins is routine in many laboratories; however, many of the obtained sequences are incomplete or can be misinterpreted when the samples contain unusual amino acids. Here we report the development of an automated peptide sequenator coupled to an electrospray-ionization (ESI) mass spectrometer (MS) that, in conjunction with minor modifications to the sequencing conditions and, in some cases, prior derivatization of amino acids, allows the detection of the phenylthiohydantoin (PTH) derivatives of a number of unusual amino acids. Using the coupled sequenator-ESI-MS system we were able to determine the complete sequence of the lantibiotic gallidermin, a partial sequence of the calcium-dependent peptide antibiotic CDA2 as well as the pool sequence of a mixture of synthetic peptides containing nonproteinogenic amino acids.

Pancreas specific protein disulfide isomerase, PDIp, is in transient contact with secretory proteins during late stages of translocation.

Protein disulfide isomerase (PDI) and an additional lumenal protein of dog pancreas microsomes were previously observed to be in transient contact with secretory proteins during late stages of their co- or posttranslational translocation into these mammalian microsomes. The second protein was characterized as a 57 kDa glycoprotein. Here we identified this glycoprotein as the canine equivalent of human PDIp, a protein which was recently described as a new protein disulfide isomerase which is highly expressed in human pancreas.

Natural ligand motifs of closely related HLA-DR4 molecules predict features of rheumatoid arthritis associated peptides.

Rheumatoid arthritis (RA), one of the most common autoimmune disorders, is believed to be mediated via. T lymphocytes and genetic studies have shown that it is strongly associated with HLA-DR4. The DR4 subtypes DR4Dw4, DR4Dw14 and DR4Dw15 represent increased risk factors for RA, whereas DR4Dw10 is not associated with the disorder.

Cathepsin L is an intracellular and extracellular protease in Paramecium tetraurelia. Purification, cloning, sequencing and specific inhibition by its expressed propeptide.

The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed.

Biochemical and molecular characterization of the extracellular esterase from Streptomyces diastatochromogenes.

An esterase of Streptomyces diastatochromogenes was purified to homogeneity from culture filtrate. The purified enzyme had a molecular mass of 30,862 +/- 5.8 Da, as determined by electrospray mass spectrometry.

Protein engineering of lantibiotics.

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply.

Isolation and characterization of genetically engineered gallidermin and epidermin analogs.

Gallidermin (Gdm) and epidermin (Epi) are highly homologous tetracyclic polypeptide antibiotics that are ribosomally synthesized by a Staphylococcus gallinarum strain and a Staphylococcus epidermidis strain, respectively. These antibiotics are secreted into media and are distinguished by the presence of the unusual amino acids lanthionine, 3-methyllanthionine, didehydrobutyrine, and S-(2-aminovinyl)-D-cysteine, which are formed by posttranslational modification. To study the substrate specificities of the modifying enzymes and to obtain variants that exhibit altered or new biological activities, we changed certain amino acids by performing site-specific mutagenesis with the Gdm and Epi structural genes (gdmA and epiA, respectively).

Integrin alpha 5 during early development of Xenopus laevis.

The full length sequence of the Xenopus integrin alpha 5 subunit is reported. Analysis of cloned cDNA fragments reveals that alternative polyadenylation of alpha 5 mRNA occurs in the embryo. Furthermore, a variant form of the alpha 5 mRNA is expressed which encodes an integrin alpha 5 subunit with a truncated cytoplasmic domain.

Supermotifs enable natural invariant chain-derived peptides to interact with many major histocompatibility complex-class II molecules.

Class II-associated invariant chain peptides (CLIPs) compete with natural allele-specific ligands for binding to several purified HLA-DR molecules. Truncation and substitution analysis showed that a minimal sequence of 13 amino acids is sufficient for excellent binding to DR17 and DR1. Hydrophobic residues at relative positions 1 and 9 (P1 and P9) which are shared among these DR-ligands, and are found to be anchored in complementary pockets by x-ray crystallography allow specific binding.

Peptide motifs of HLA-B51, -B52 and -B78 molecules, and implications for Behćet's disease.

Here we report peptide motifs of five HLA-B molecules, B*5101, B*5102, B*5103 B*5201 and B*7801. Motifs were obtained by pool sequencing of natural ligands eluted from the respective molecules expressed in C1R cells upon transfection. A number of individual ligands that could be sequenced confirmed the motifs.

Distribution of parasite cysteine proteinases in lesions of mice infected with Leishmania mexicana amastigotes.

It is well established that Leishmania mexicana amastigotes contain large amounts of cysteine proteinases in their extended lysosomes. In this study it is shown that the cell-free supernatant of homogenized lesion tissue from infected mice contains large amounts of acid proteinases. The majority of this enzymatic activity also corresponds to cysteine proteinases from L.

A 16mer peptide of the human autoantigen calreticulin is a most prominent HLA-DR4Dw4-associated self-peptide.

The human Ca(2+)-binding (storage) protein calreticulin, located in the lumen of the endoplasmic reticulum, is proposed to play a role as autoantigen: anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis (calreticulin shows a high sequence homology to the Onchocerca volvulus antigen RAL-1). Here we present sequencing data of a HLA-DR4Dw4-associated calreticulin peptide fragment, Cal(295-310), purified from a DR4Dw4 self-peptide pool. Cal(295-310) proved to be one of three commonest self-peptides associated with DR4Dw4 molecules that were isolated from the EBV-transformed B-cell line BSM (DR4Dw4, DRw53).

Analysis of allele-specific contact sites of natural HLA-DR17 ligands.

The sequence motif of peptide ligands naturally associated with DR17 has indicated conserved residues at the relative positions P1-P4-P6-P 8.9 or 10. Eight naturally processed DR17 ligands were synthesized to study the role of conserved residues in DR17 binding.

Analysis of a naturally occurring HLA class I-restricted viral epitope.

A previously described nonapeptide sequence motif for antigens recognized by T cells in the context of the human major histocompatibility complex (MHC) molecule HLA-A2.1 was used to identify the natural epitope of influenza A virus matrix protein. We show here that the peptide with the sequence GILGFVFTL is the synthetic analogue of the natural epitope by demonstrating the presence of the corresponding peptide on MHC molecules of virus-infected cells.

Mass spectroscopic analysis of a novel enzymatic reaction. Oxidative decarboxylation of the lantibiotic precursor peptide EpiA catalyzed by the flavoprotein EpiD.

The epidermin biosynthetic reaction between the flavoprotein EpiD and the precursor peptide EpiA was investigated by reversed-phase chromatography and ion spray mass spectrometry. Several products with molecular masses 46 and 104 Da less than that of EpiA were observed; these results were confirmed by using an MBP-EpiD fusion protein as enzyme and the mutant peptides EpiAR-1Q and K-EpiA as substrates. The reaction was inhibited by Zn2+ ions.