Identification of a 68 kDa protein which copurifies with type-1 protein phosphatase as albumin

AbstractProteins of 60–70 kDa copurify with some preparations of type-1 or type-2 phosphatases. In our system chromatography on polylysine-Affi-Gel 10 separates a 68 kDa protein from rabbit muscle glycogen particle phosphorylase phosphatase. The separation affects neither the activity nor the size of the phosphatase. The 68 kDa protein, although pure by SDS gel electrophoresis criteria, still displays phosphatase activity of approx. 6–8 Umg. However, rechromatography either on Bio-Gel A-0.5 m or on Blue Sepharose CL-6B followed by gel filtration shows that the activity is due to a contamination with phosphatases of type 1 and type 2, displaying a molecular mass of 35 kDa, which can be totally removed from the 68 kDa protein. The amino acid composition of the 68 kDa protein is identical to that of rabbit serum albumin, within the limits of variation of the method. Furthermore, the sequence of the 38 N-terminal amino acids is the same in the isolated 68 kDa protein and in rabbit serum albumin

Proteome analyses of hepatocellular carcinoma

Proteomics has evolved into a powerful and widely used bioanalytical technique in the study of cancer, especially hepatocellular carcinoma (HCC). In this review, we provide an up to date overview of feasible proteome-analytical techniques for clinical questions. In addition, we present a broad summary of proteomic studies of HCC utilizing various technical approaches for the analysis of samples derived from diverse sources like HCC cell lines, animal models, human tissue and body fluids

Characterisation of the autoinmune antibody repertoire of Parkinson's disease patients by systematic screening of protein arrays

Comunicaciones a congreso

Cooperation of translocase complexes in mitochondrial protein import

Most mitochondrial proteins are synthesized in the cytosol and imported into one of the four mitochondrial compartments: outer membrane, intermembrane space, inner membrane, and matrix. Each compartment contains protein complexes that interact with precursor proteins and promote their transport. These translocase complexes do not act as independent units but cooperate with each other and further membrane complexes in a dynamic manner. We propose that a regulated coupling of translocases is important for the coordination of preprotein translocation and efficient sorting to intramitochondrial compartments

Intricate Crosstalk Between Lipopolysaccharide, Phospholipid and Fatty Acid Metabolism in Escherichia coli Modulates Proteolysis of LpxC

Lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria provide the first line of defense against antibiotics and other harmful compounds. LPS biosynthesis critically depends on LpxC catalyzing the first committed enzyme in this process. In Escherichia coli, the cellular concentration of LpxC is adjusted in a growth rate-dependent manner by the FtsH protease making sure that LPS biosynthesis is coordinated with the cellular demand. As a result, LpxC is stable in fast-growing cells and prone to degradation in slow-growing cells. One of the factors involved in this process is the alarmone guanosine tetraphosphate (ppGpp) but previous studies suggested the involvement of yet unknown factors in LpxC degradation. We established a quantitative proteomics approach aiming at the identification of proteins that are associated with LpxC and/or FtsH at high or low growth rates. The identification of known LpxC and FtsH interactors validated our approach. A number of proteins involved in fatty acid biosynthesis and degradation, including the central regulator FadR, were found in the LpxC and/or FtsH interactomes. Another protein associated with LpxC and FtsH was WaaH, a LPS-modifying enzyme. When overproduced, several members of the LpxC/FtsH interactomes were able to modulate LpxC proteolysis. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS production by involving multiple enzymes in fatty acid metabolism, phospholipid biosynthesis and LPS modification

Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing

AbstractTwo phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzymic activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids. The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358–386, 862–882) are detectable. Furthermore, a helix loop helix motif (amino acids 716–729) as well as two nuclear localization signals (amino acids 838–854, 345–349) indicate the presence of the type 3 isoform in the nucleus

Identification of Ser-1275 and Ser-1309 as autophosphorylation sites of the insulin receptor 1This paper is dedicated to Prof. Günter Legler on the occasion of his 70th birthday.1

AbstractWe have identified Ser-1275 and Ser-1309 as novel serine autophosphorylation sites by direct sequencing of HPLC-purified tryptic phosphopeptides of the histidine-tagged insulin receptor kinase IRKD-HIS. The corresponding peptides (Ser-1275, amino acids 1272–1292; Ser-1309, amino acids 1305–1313) have been detected in the HPLC profiles of both the soluble kinase IRKD, which contains the entire cytoplasmic domain of the insulin receptor β-subunit, and the insulin receptor purified from human placenta. In contrast, a kinase negative mutant, IRKD-K1018A, did not undergo phosphorylation at either the tyrosine or serine residues, strongly suggesting that insulin receptor kinase has an intrinsic activity to autophosphorylate serine residues

The in vivo phosphorylation sites of bovine αB-crystallin

AbstractPhosphate content determinations established that in αB-crystallin two phosphate groups can be present in vivo in bovine lenses. Comparison of tryptic digests of phosphorylated and unphosphorylated αB chains, revealed the location of the two phosphorylation sites in tryptic peptides T2 and T3. Thermolytic digestion and gas-phase sequencing demonstrated that Ser-19 and Ser-45 are the in vivo phosphorylation sites of bovine αB-crystallin. This pattern of phosphorylation differs from the previously reported in vitro obtained results

Unintended spread of a biosafety level 2 recombinant retrovirus

<p>Abstract</p> <p>Background</p> <p>Contamination of vertebrate cell lines with animal retroviruses has been documented repeatedly before. Although such viral contaminants can be easily identified with high sensitivity by PCR, it is impossible to screen for all potential contaminants. Therefore, we explored two novel methods to identify viral contaminations in cell lines without prior knowledge of the kind of contaminant.</p> <p>Results</p> <p>The first hint for the presence of contaminating retroviruses in one of our cell lines was obtained by electron microscopy of exosome-like vesicles released from the supernatants of transfected 293T cells. Random amplification of particle associated RNAs (PAN-PCR) from supernatant of contaminated 293T cells and sequencing of the amplicons revealed several nucleotide sequences showing highest similarity to either murine leukemia virus (MuLV) or squirrel monkey retrovirus (SMRV). Subsequent mass spectrometry analysis confirmed our findings, since we could identify several peptide sequences originating from monkey and murine retroviral proteins. Quantitative PCRs were established for both viruses to test currently cultured cell lines as well as liquid nitrogen frozen cell stocks. Gene fragments for both viruses could be detected in a broad range of permissive cell lines from multiple species. Furthermore, experimental infections of cells negative for these viruses showed that both viruses replicate rapidly to high loads. We decided to further analyze the genomic sequence of the MuLV-like contaminant virus. Surprisingly it was neither identical to MuLV nor to the novel xenotropic MuLV related retrovirus (XMRV) but showed 99% identity to a synthetic retrovirus which was engineered in the 1980s.</p> <p>Conclusion</p> <p>The high degree of nucleotide identity suggests unintended spread of a biosafety level 2 recombinant virus, which could also affect the risk assessment of gene-modified organisms released from contaminated cell cultures. The study further indicates that both mass spectrometry and PAN-PCR are powerful methods to identify viral contaminations in cell lines without prior knowledge of the kind of contaminant. Both methods might be useful tools for testing cell lines before using them for critical purposes.</p