Cdc45-induced loading of human RPA onto single-stranded DNA.

Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA.

Human DNA polymerase α interacts with mismatch repair proteins MSH2 and MSH6.

High fidelity of genome duplication is ensured by cooperation of polymerase proofreading and mismatch repair (MMR) activities. Here, we show that human mismatch recognizing proteins MutS homolog 2 (MSH2) and MSH6 copurify and interact with replicative Pol α. This enzyme also is the replicative primase and replicates DNA with poor fidelity.

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci.

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase.

Caspase-3 and caspase-6 cleave STAT1 in leukemic cells.

Signal Transducer and Activator of Transcription-1 (STAT1) is phosphorylated upon interferon (IFN) stimulation, which can restrict cell proliferation and survival. Nevertheless, in some cancers STAT1 can act in an anti-apoptotic manner. Moreover, certain malignancies are characterized by the overexpression and constitutive activation of STAT1.

Identification of prolyl oligopeptidase as a cyclosporine-sensitive protease by screening of mouse liver extracts.

Cyclosporine A (CsA) is a cyclic undecapeptide well known for its ability to prevent rejection episodes after organ transplantation via gain-of-function. Therefore, biomedical studies on CsA have been focused on both immunosuppressive properties and binding to the biocatalytically-active immune receptors, the cyclophilins. Much less attention has been spent on effects of cyclosporines on the biological function of other proteins.

Proteomic identification of PSF and p54(nrb) as TopBP1-interacting proteins.

TopBP1 is a BRCT domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1-interacting proteins by co-immunoprecipitation assays and LC-ESI-MS-analyses.

Synthesis and functional characterization of tridegin and its analogues: inhibitors and substrates of factor XIIIa.

Tridegin, a 66-mer peptide isolated from the leech Haementeria ghilianii, is a potent inhibitor of the coagulation factor XIIIa. This paper describes the chemical synthesis of tridegin by two different strategies--solid-phase assembly and native chemical ligation--both followed by oxidation in solution phase. Tridegin and truncated analogues were examined for their activity and revealed a particular importance of the C-terminal region of the parent peptide.

Determination of hemin-binding characteristics of proteins by a combinatorial peptide library approach.

Studies of the binding of heme/hemin to proteins or peptides have recently intensified as it became evident that heme serves not only as a prosthetic group, but also as a regulator and effector molecule interacting with transmembrane and cytoplasmic proteins. The iron-ion-containing heme group can associate with these proteins in different ways, with the amino acids Cys, His, and Tyr allowing individual modes of binding. Strong coordinate-covalent binding, such as in cytochrome c, is known, and reversible attachment is also discussed.

Molecular and biochemical properties of the S-layer protein from the wine bacterium Lactobacillus hilgardii B706.

Different strains of the genus Lactobacillus can be regularly isolated from must and wine samples. By various physiological activities, they can improve or reduce the wine quality. Lactobacillus hilgardii that is known to survive under harsh wine conditions is classified as a spoilage bacterium, e.

Cervimycin C resistance in Bacillus subtilis is due to a promoter up-mutation and increased mRNA stability of the constitutive ABC-transporter gene bmrA.

Two independent cervimycin C (CmC)-resistant clones of Bacillus subtilis were identified, each carrying two mutations in the intergenic region preceding the ABC transporter gene bmrA. In the double mutant, real-time PCR revealed an increased amount of bmrA mRNA with increased stability. Accordingly, isolation of membrane proteins yielded a strong band at 64 kDa corresponding to BmrA.

Structure and biochemical analysis of the heparin-induced E1 dimer of the amyloid precursor protein.

The amyloid precursor protein (APP) is the key player in Alzheimer's disease pathology, yet APP and its analogues are also essential for neuronal development and cell homeostasis in mammals. We have determined the crystal structure of the entire N-terminal APP-E1 domain consisting of the growth factor like and the copper binding domains at 2.7-A resolution and show that E1 functions as a rigid functional entity.

Consequences of monocarboxylate transporter 8 deficiency for renal transport and metabolism of thyroid hormones in mice.

Patients carrying inactivating mutations in the gene encoding the thyroid hormone transporting monocarboxylate transporter (MCT)-8 suffer from a severe form of psychomotor retardation and exhibit abnormal serum thyroid hormone levels. The thyroidal phenotype characterized by high-serum T(3) and low-serum T(4) levels is also found in mice mutants deficient in MCT8 although the cause of these abnormalities is still unknown. Here we describe the consequences of MCT8 deficiency for renal thyroid hormone transport, metabolism, and function by studying MCT8 null mice and wild-type littermates.

Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B.

Protein-tyrosine phosphatases (PTPs) are regulated by reversible inactivating oxidation of the catalytic-site cysteine. We have previously shown that reversible oxidation upon UVA irradiation is followed by calpain-mediated PTP degradation. Here, we address the mechanism of regulated cleavage and the physiological function of PTP degradation.

The ether-cleaving methyltransferase system of the strict anaerobe Acetobacterium dehalogenans: analysis and expression of the encoding genes.

Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A.

PI3Kgamma controls oxidative bursts in neutrophils via interactions with PKCalpha and p47phox.

Neutrophils release reactive oxygen species (ROS) as part of the innate inflammatory immune response. Phosphoinositide 3-kinase gamma (PI3Kgamma), which is induced by the bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP), has been identified as an essential intracellular mediator of ROS production. However, the complex signalling reactions that link PI3Kgamma with ROS synthesis by NADPH oxidase have not yet been described in detail.

A novel Dps-type protein from insect gut bacteria catalyses hydrolysis and synthesis of N-acyl amino acids.

A novel type of a microbial N-acyl amino acid hydrolase (AAH) from insect gut bacteria was purified, cloned and functionally characterized. The enzyme was obtained from Microbacterium arborescens SE14 isolated from the foregut of larvae of the generalist herbivore Spodoptera exigua. The substrates of AAH are N-acyl-glutamines previously reported to elicit plant defence reactions after introduction into the leaf during feeding.

The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli.

Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown.

Molecular organization of selected prokaryotic S-layer proteins.

Regular crystalline surface layers (S-layers) are widespread among prokaryotes and probably represent the earliest cell wall structures. S-layer genes have been found in approximately 400 different species of the prokaryotic domains bacteria and archaea. S-layers usually consist of a single (glyco-)protein species with molecular masses ranging from about 40 to 200 kDa that form lattices of oblique, tetragonal, or hexagonal architecture.

Characterization of the protein components of Nephila clavipes dragline silk.

Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads.

N-acetyl-D-galactosamine/N-acetyl-D-glucosamine--recognizing lectin from the snail Cepaea hortensis: purification, chemical characterization, cloning and expression in E. coli.

From the albumin gland of the snail Cepaea hortensis we isolated and characterized a new N-acetyl-D-galactosamine/N-acetyl-D-glucosamine (GalNAc/GlcNAc) specific lectin (CHA-II) which was purified by a combination of affinity chromatography on GalNAc-agarose and gel filtration. The purified native lectin was found to be a multimeric protein, as revealed by SDS-PAGE and MALDI-TOF analysis. In SDS-PAGE the denatured and reduced lectin showed two bands of molecular masses with 17 and 15.

Partial proteolysis of simian virus 40 T antigen reveals intramolecular contacts between domains and conformation changes upon hexamer assembly.

Simian virus 40 large tumor antigen (Tag) is a multi-functional viral protein that binds specifically to SV40 origin DNA, serves as the replicative DNA helicase, and orchestrates the assembly and operation of the viral replisome. Tag associated with Mg-ATP forms hexamers and, in the presence of SV40 origin DNA, double hexamers. Limited tryptic digestion of monomeric Tag revealed three major stable structural domains.

Cloning and expression of a sialic acid-binding lectin from the snail Cepaea hortensis.

Highly degenerated gene-specific oligonucleotide primers (GSPs) were constructed from the amino acid sequence of tryptic fragments produced from the purified sialic acid-specific lectin of the garden snail Cepaea hortensis. From the albumin glands, the total RNA or the mRNA was prepared. Combination of a universal primer with the GSPs delivered gene-specific fragments of about 650, 620 and 280 bp by polymerase chain reaction (PCR).

DNA-dependent protein kinase (DNA-PK) phosphorylates nuclear DNA helicase II/RNA helicase A and hnRNP proteins in an RNA-dependent manner.

An RNA-dependent association of Ku antigen with nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), was found in nuclear extracts of HeLa cells by immunoprecipitation and by gel filtration chromatography. Both Ku antigen and NDH II were associated with hnRNP complexes. Two-dimensional gel electrophoresis showed that Ku antigen was most abundantly associated with hnRNP C, K, J, H and F, but apparently not with others, such as hnRNP A1.

Human replication protein A. The C-terminal RPA70 and the central RPA32 domains are involved in the interactions with the 3'-end of a primer-template DNA.

Although the mechanical aspects of the single-stranded DNA (ssDNA) binding activity of human replication protein A (RPA) have been extensively studied, only limited information is available about its interaction with other physiologically relevant DNA structures. RPA interacts with partial DNA duplexes that resemble DNA intermediates found in the processes of DNA replication and DNA repair. Limited proteolysis of RPA showed that RPA associated with ssDNA is less protected against proteases than RPA bound to a partial duplex DNA containing a 5'-protruding tail that had the same length as the ssDNA.