Identification and characterization of FliY, a novel component of the Bacillus subtilis flagellar switch complex

The Bacillus subtilis gene encoding FliY has been cloned and sequenced. The gene encodes a 379-amino-acid protein with a predicted molecular mass of 41,054 daltons. FliY is partly homologous to the Escherichia coli and Salmonella typhimurium switch proteins FliM and FliN. The N-terminus of FliY has 33% identity with the first 122 amino acids of FliM, whereas the C-terminus of FliY has 52% identity with the last 30 amino acids of FliN. The middle 60% of FliY is not significantly homologous to either of the proteins. A fliY::cat null mutant has no flagella. Motility can be restored to the mutant by expression of fliY from a plasmid, although chemotaxis is still defective since the strain exhibits smooth swimming behaviour. fliY::cat is in the cheD complementation group. One of the cheD point mutants does not switch although the population grown from a single cell has both smooth swimming and tumbling bacteria, implying that the switch is locked. Expression of fliY in wild-type B. subtilis makes the cells more smooth-swimming but does not appear to affect chemotaxis. Expression of fliY in wild-type S. typhimurium severely inhibits chemotaxis and also makes the cells smooth swimming. Expression in a non-motile S. typhimurium fliN mutant restores motility but not chemotaxis, although expression in a non-motile E. coli fliM mutant does not restore motility. The homology, multiple phenotypes, and interspecies complementation suggest that FliY forms part of the B. subtilis switch complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of Gemin7, a novel component of the survival of motor neuron complex

The survival of motor neurons (SMN) protein is the product of the gene mutated or deleted in the neurodegenerative disease, spinal muscular atrophy. SMN is part of a large macromolecular complex that also contains Gemin2, Gemin3, Gemin4, Gemin5, and Gemin6. The SMN complex functions in the assembly of spliceosomal small nuclear ribonucleoproteins and probably other ribonucleoprotein particles. We have identified a novel protein component of the SMN complex termed Gemin7 using native purified SMN complexes and peptide sequencing by mass spectrometry. Coimmunoprecipitation and immunolocalization experiments demonstrate that Gemin7 is a component of the SMN complex and colocalizes with SMN in the cytoplasm and in gems. Binding experiments show that Gemin7 interacts directly with SMN and Gemin6 and mediates the association of Gemin6 with the SMN complex. The amino acid sequence of Gemin7 does not contain any recognizable motifs with the exception of several arginine and glycine repeats that are necessary for its interaction with SMN. Moreover, Gemin7 interacts with several Sm proteins of spliceosomal small nuclear ribonucleoproteins, in particular, with SmE. With the identification of Gemin7, the inventory of the core components of the SMN complex appears essentially complete.     

Identification and characterization of a novel component of the cornified envelope, cornifelin

Psoriasis is recognized as a chronic inflammatory disease characterized by epidermal hyperproliferation. To identify psoriasis-related genes, we compared the mRNA populations of normal and psoriatic skin. We identified one gene, designated as cornifelin, which showed increased expression in psoriatic skin. Human cornifelin contains 112 amino acids and is expressed in the uterus, cervix, and skin. In situ hybridization analysis demonstrated the presence of human cornifelin in the granular cell layer of the epidermis. To investigate the function of cornifelin, we established a transgenic mouse line overexpressing human cornifelin. Using these mice, we have shown that cornifelin is directly or indirectly cross-linked to at least two other cornified envelope proteins, loricrin and involucrin, in vivo. Overexpression of human cornifelin correlated with decreased loricrin expression and increased involucrin expression in the transgenic mouse. However, abnormality of epidermal differentiation was not observed in the transgenic mouse.     

Biochemical characterization of the minichromosome maintenance protein from the archaeon Thermoplasma acidophilum

Minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. Studies have shown that the MCM complex from the thermoacidophilic euryarchaeon Thermoplasma acidophilum (TaMCM) has some properties not reported in other archaeal MCM helicases. Here, the biochemical properties of the TaMCM are studied. The protein binds single-stranded DNA, has DNA-dependent ATPase activity and ATP-dependent 3′ → 5′ helicase activity. The optimal helicase conditions with regard to temperature, pH and salinity are similar to the intracellular conditions in T. acidophilum. It is also found that about 1,000 molecules of TaMCM are present per actively growing cell. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and characterization of a novel human collectin CL-K1

Collectins are a family of C-type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host-defense. Here we report the cloning and characterization of a novel collectin CL-K1. RT-PCR analyses showed CL-K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL-K1 cDNA expressing CHO cells revealed that CL-K1 is expressed as a secreted protein. CL-K1 is found in blood by immunoblotting and partial amino acid analyses. CL-K1 showed Ca(2+)-dependent sugar binding activity of fucose and weakly mannose but not N-acetyl-galactosamine, N-acetyl-glucosamine, or maltose, though mannose-binding lectin (MBL) containing similar amino acid motif. CL-K1 can recognize specially several bacterial saccharides due to specific sugar-binding character. Elucidation of the role of two ancestor collectins of CL-K1 and CL-L1 could lead to see the biological function of collectin family.     

Identification of a novel non-structural maintenance of chromosomes (SMC) component of the SMC5-SMC6 complex involved in DNA repair

Structural maintenance of chromosomes (SMC) proteins play central roles in chromosome organization and dynamics. They have been classified into six subtypes, termed SMC1 to SMC6, and function as heterodimer components of large protein complexes that also include several non-SMC proteins. The SMC2-SMC4 and SMC1-SMC3 complexes are also known as condensin and cohesin, respectively, but the recently identified SMC5 and SMC6 complex is less well characterized. Here, we report that NSE1 from Saccharomyces cerevisiae encodes a novel non-SMC component of the SMC5(Yol034wp)-SMC6(Rhc18p) complex corresponding to the 2-3-MDa molecular mass. Nse1p is essential for cell proliferation and localizes primarily in the nucleus. nse1 mutants are highly sensitive to DNA-damaging treatments and exhibit abnormal cellular morphologies, suggesting aberrant mitosis as a terminal morphological phenotype. These results are consistent with the reported features of the Schizosaccharomyces pombe SMC6 gene, rad18, which is thought to be involved in recombinational DNA repair. We conclude that Nse1p and the SMC5-SMC6 heterodimer together form a high molecular mass complex that is conserved in eukaryotes and required for both DNA repair and proliferation.     

Identification and characterization of a novel monoamine transporter in the human brain

Precise control of monoamine neurotransmitter levels in the extracellular fluids of the brain is critical in maintaining efficient and robust neurotransmission. High affinity transporters in the solute carrier SLC6A family function in removing monoamines from the neurosynaptic cleft. Emerging evidence suggests that these transporters are only one part of a system of transporters that work in concert to maintain brain homeostasis of monoamines. Here we report the cloning and characterization of a new human plasma membrane monoamine transporter, PMAT. The PMAT cDNA encodes a protein of 530 amino acid residues with 10-12 transmembrane segments. PMAT is not homologous to known neurotransmitter transporters but exhibits low homology to members of the equilibrative nucleoside transporter family. When expressed in Madin-Darby canine kidney cells and Xenopus laevis oocytes, PMAT efficiently transports serotonin (K(m) = 114 mum), dopamine (K(m) = 329 mum), and the neurotoxin 1-methyl-4-phenylpyridinium (K(m) = 33 mum). In contrast, there is no significant interaction of PMAT with nucleosides or nucleobases. PMAT-mediated monoamine transport does not require Na(+) or Cl(-) but appears to be sensitive to changes in membrane potential. Northern blot analysis showed that PMAT is predominantly expressed in the human brain and widely distributed in the central nervous system. These studies demonstrate that PMAT may be a novel low affinity transporter for biogenic amines, which, under certain conditions, might supplement the role of the high affinity transporters in the brain.     

Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe

Minichromosome maintenance (Mcm) proteins 2-7 are highly conserved in eukaryotes and play an essential role in DNA replication. Here, we describe the reconstitution of the various complexes of the Mcm proteins of Schizosaccharomyces pombe using the baculovirus expression system. The simultaneous expression of all six of the Mcm proteins, as well as different combinations of these proteins, yielded several stable complexes that included the heterohexamer of Mcm2/3/4/5/6/7, the Mcm2/4/6/7 heterotetramer, the dimer of the Mcm4/6/7 heterotrimer, and the Mcm3/5 heterodimer. The purification and characterization of the biochemical properties of these complexes showed that only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities. Consistent with these results, the interaction of either Mcm2 or Mcm3/5 with the Mcm4/6/7 complex resulted in the disassembly of the dimeric complex of Mcm4/6/7 and the loss of DNA helicase activity. These results suggest that the Mcm4/6/7 complex is a catalytic core of the Mcm complex and that Mcm2 and Mcm3/5 may be involved in the regulation of the activity of this complex.     

人端粒保护蛋白hPot1的一种新选择性剪接体的克隆及鉴定

hPot1是端粒单链结合蛋白,在维持染色体末端的稳定性中发挥着重要作用。从前列腺癌细胞C4-2中提取总RNA,以反转录得到的cDNA为模板,扩增全长的hPot1 cDNA,发现hPot1基因的一种新的剪接形式。这种新的剪接形式缺失了野生型hPot1基因的第2个外显子,并且造成了读码框架的改变,使翻译提前终止,表达出一段有45个氨基酸残基的短肽。进一步检测表明,这一hPot1 mRNA新剪接体广泛存在于多种组织来源的细胞中,提示这一剪接形式可能是细胞调控hPot1功能的一种调节机制。     

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.     

Biochemical characterization of the Methanothermobacter thermautotrophicus minichromosome maintenance (MCM) helicase N-terminal domains

Minichromosome maintenance helicases are ring-shaped complexes that play an essential role in archaeal and eukaryal DNA replication by separating the two strands of chromosomal DNA to provide the single-stranded substrate for the replicative polymerases. For the archaeal protein it was shown that the N-terminal portion of the protein, which is composed of domains A, B, and C, is involved in multimer formation and single-stranded DNA binding and may also play a role in regulating the helicase activity. Here, a detailed biochemical characterization of the N-terminal region of the Methanothermobacter thermautotrophicus minichromosome maintenance helicase is described. Using biochemical and biophysical analyses it is shown that domain C of the N-terminal portion, located adjacent to the helicase catalytic domains, is required for protein multimerization and that domain B is the main contact region with single-stranded DNA. It is also shown that although oligomerization is not essential for single-stranded DNA binding and ATPase activity, the presence of domain C is essential for helicase activity.     

Cdt1 forms a complex with the minichromosome maintenance protein (MCM) and activates its helicase activity

Mcm4/6/7 forms a complex possessing DNA helicase activity, suggesting that Mcm may be a central component for the replicative helicase. Although Cdt1 is known to be essential for loading of Mcm onto the chromatin, its precise role in pre-RC formation and replication initiation is unknown. Using purified proteins, we show that Cdt1 forms a complex with Mcm4/6/7, Mcm2/3/4/5/6/7, and Mcm2/4/6/7 in glycerol gradient fractionation through interaction with Mcm2 and Mcm4/6. In the glycerol gradient fractionation, Mcm4/6/7-Cdt1 forms a complex (speculated to be a (Mcm4/6/7)2-Cdt13 assembly) in the presence of ATP, which is significantly larger than the Mcm4/6/7-Cdt1 complex generated in its absence. Furthermore, DNA binding and helicase activities of Mcm4/6/7 are significantly stimulated by Cdt1 protein in vitro. We generated a Cdt1 mutant, which fails to stimulate DNA binding and helicase activities of Mcm4/6/7. This mutant Cdt1 showed reduced interaction with Mcm and is deficient in the formation of a high molecular weight complex with Mcm. Thus, a productive interaction between Cdt1 and MCM appears to be essential for efficient loading of MCM onto template DNA, as well as for the efficient unwinding reaction.     

Identification and characterization of a novel agonistic anti-DR4 human monoclonal antibody

The tumor necrosis factor-related apoptosis–inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been established as promising targets for cancer treatment. Therapeutics targeting TRAIL and its receptors are not only effective in killing many types of tumors, but they also synergize with traditional therapies and show efficacy against tumors that are otherwise resistant to conventional treatments. We describe here the identification and characterization of two human monoclonal antibodies, m921 and m922, that are specific for human DR4. Both antibodies competed with TRAIL for binding to DR4, but only m921 recognized cell surface-associated DR4 and inhibited the growth of ST486 cells. This antibody may have potential for further development as a candidate therapeutic and research tool.     

Identification and characterization of a novel agonistic anti-DR4 human monoclonal antibody

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been established as promising targets for cancer treatment. Therapeutics targeting TRAIL and its receptors are not only effective in killing many types of tumors, but they also synergize with traditional therapies and show efficacy against tumors that are otherwise resistant to conventional treatments. We describe here the identification and characterization of two human monoclonal antibodies, m921 and m922, that are specific for human DR4. Both antibodies competed with TRAIL for binding to DR4, but only m921 recognized cell surface-associated DR4 and inhibited the growth of ST486 cells. This antibody may have potential for further development as a candidate therapeutic and research tool.Key words: DR4, TRAIL, lymphoma, therapeutic antibody     

Complete in vitro reconstitution of adeno-associated virus DNA replication requires the minichromosome maintenance complex proteins

Adeno-associated virus (AAV) replicates its DNA exclusively by a leading-strand DNA replication mechanism and requires coinfection with a helper virus, such as adenovirus, to achieve a productive infection. In previous work, we described an in vitro AAV replication assay that required the AAV terminal repeats (the origins for DNA replication), the AAV Rep protein (the origin binding protein), and an adenovirus-infected crude extract. Fractionation of these crude extracts identified replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and polymerase δ as cellular enzymes that were essential for AAV DNA replication in vitro. Here we identify the remaining factor that is necessary as the minichromosome maintenance (MCM) complex, a cellular helicase complex that is believed to be the replicative helicase for eukaryotic chromosomes. Thus, polymerase δ, RFC, PCNA, and the MCM complex, along with the virally encoded Rep protein, constitute the minimal protein complexes required to reconstitute efficient AAV DNA replication in vitro. Interfering RNAs targeted to MCM and polymerase δ inhibited AAV DNA replication in vivo, suggesting that one or more components of the MCM complex and polymerase δ play an essential role in AAV DNA replication in vivo as well as in vitro. Our reconstituted in vitro DNA replication system is consistent with the current genetic information about AAV DNA replication. The use of highly conserved cellular replication enzymes may explain why AAV is capable of productive infection in a wide variety of species with several different families of helper viruses.