Crystal structure and mutagenic analysis of a bacteriocin immunity protein,Mun-im

Bacteriocin-producing lactic acid bacteria (LAB) possess a self-protection factor, which is generally called an immunity protein. In this study, we determine the crystal structure of an immunity protein, designated Mun-im, which was classified into subgroup B immunity proteins for class IIa bacteriocins. The Mun-im protein takes a left-turning antiparallel four-helix bundle structure with the flexible N- and C-terminal parts. Although the amino acid sequences of the subgroup B immunity proteins are distinguished from those of the subgroup A, the core structure of Mun-im is well-superimposed with that of the subgroup A immunity protein, EntA-im, and the C-terminus of both proteins is flexible. However, the C-terminus of Mun-im is obviously shorter than that of the subgroup A. We found through mutagenic study of Mun-im that the C-terminus and the K86 residue on the helix 4 in the immunity protein molecule are important for expression of the immunity activity on the subgroup B immunity proteins.     

CrxOS maintains the self-renewal capacity of murine embryonic stem cells

Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.     

Hematopoietic stem cell aging is associated with functional decline and delayed cell cycle progression

The molecular mechanisms underlying hematopoietic stem cell (HSC) aging remain to be elucidated. In this study, we investigated age-related changes in the functional and phenotypic properties of murine HSCs. Consistent with previous studies, we found that the number and frequency of CD34^−/lowc-Kit⁺Sca-1⁺lineage marker⁻ (CD34⁻KSL) cells, a highly enriched HSC population, significantly increased in old mice, though their repopulating ability was reduced. Continuous bromodeoxyuridine labeling revealed a significant delay in the cell cycle progression of CD34⁻KSL cells in old mice. This delay was also observed in young recipients transplanted with whole bone marrow cells from old mice. When cultured in vitro, CD34⁻KSL cells from old mice showed a greater capacity to give rise to primitive CD48⁻KSL cells with reduced HSC activity. Gene expression profiling identified age-related changes in the expression of several cell cycle regulatory genes, including p21/Cdkn1a and p18/Cdkn2c. These results support the notion that HSC aging is largely regulated by an intrinsic genetic program.     

The effect of the side chain length of Asp and Glu on coordination structure of Cu2+ in a de novo designed protein

Metal ions in proteins are important not only for the formation of the proper structures but also for various biological activities. For biological functions such as hydrolysis and oxidation, metal ions often adopt unusual coordination structures. We constructed a stable scaffold for metal binding to create distorted metal coordination structures. A stable four stranded α‐helical coiled‐coil structure was used as the scaffold, and the metal binding site was in the cavity created at the center of the structure. Two His residues and one Asp or Glu residue were used to coordinate the metal ions, AM2D and AM2E, respectively. Cu²⁺ bound to AM2D with an equatorial planar coordination structure with two His, one Asp, and H₂O as detected by electron spin resonance and UV spectral analyzes. On the other hand, Cu²⁺ had a slightly distorted square planar structure when it bound two His and Glu in AM2E, due to the longer side‐chain of the Glu residue as compared to the Asp residue. Computational analysis also supported the distorted coordination structure of Cu²⁺ in AM2E. This construct should be useful to create various coordinations of metal ions for catalytic functions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 907–916, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Isolation and Characterization of Patient-derived, Toxic, High Mass Amyloid ��-Protein (A��) Assembly from Alzheimer Disease Brains

Amyloid β-protein (Aβ) assemblies are thought to play primary roles in Alzheimer disease (AD). They are considered to acquire surface tertiary structures, not present in physiologic monomers, that are responsible for exerting toxicity, probably through abnormal interactions with their target(s). Therefore, Aβ assemblies having distinct surface tertiary structures should cause neurotoxicity through distinct mechanisms. Aiming to clarify the molecular basis of neuronal loss, which is a central phenotype in neurodegenerative diseases such as AD, we report here the selective immunoisolation of neurotoxic 10–15-nm spherical Aβ assemblies termed native amylospheroids (native ASPDs) from AD and dementia with Lewy bodies brains, using ASPD tertiary structure-dependent antibodies. In AD patients, the amount of native ASPDs was correlated with the pathologic severity of disease. Native ASPDs are anti-pan oligomer A11 antibody-negative, high mass (>100 kDa) assemblies that induce degeneration particularly of mature neurons, including those of human origin, in vitro. Importantly, their immunospecificity strongly suggests that native ASPDs have a distinct surface tertiary structure from other reported assemblies such as dimers, Aβ-derived diffusible ligands, and A11-positive assemblies. Only ASPD tertiary structure-dependent antibodies could block ASPD-induced neurodegeneration. ASPDs bind presynaptic target(s) on mature neurons and have a mode of toxicity different from those of other assemblies, which have been reported to exert their toxicity through binding postsynaptic targets and probably perturbing glutamatergic synaptic transmission. Thus, our findings indicate that native ASPDs with a distinct toxic surface induce neuronal loss through a different mechanism from other Aβ assemblies.     

Structural Basis for the Antiproliferative Activity of the Tob-hCaf1 Complex

The Tob/BTG family is a group of antiproliferative proteins containing two highly homologous regions, Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D) family of deadenylases and is a component of the CCR4-Not deadenylase complex. Here we determined the crystal structure of the complex of the N-terminal region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A and Box B of Tob. Cell growth assays using both wild-type and mutant proteins revealed that deadenylase activity of Caf1 is not critical but complex formation is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers several factors at its C-terminal region, such as poly(A)-binding proteins, to exert antiproliferative activity.The Tob/BTG family (also called the APRO family) is a group of antiproliferative proteins (1, 2) consisting of Tob (3), Tob2 (4), BTG1 (5), BTG2/Tis21/PC3 (6-8), PC3B (9), and ANA/BTG3 (10, 11) in mammalian cells, {"type":"entrez-nucleotide","attrs":{"text":"AF177464","term_id":"5916227","term_text":"AF177464"}}AF177464 in Drosophila, and FOG-3 in Caenorhabditis elegans (12). A recent genome project reported that the BTG/Tob family protein had already existed in Choanoflagellida Monosiga brevicollis MX1. The N-terminal region of the Tob/BTG family proteins is conserved and includes two highly homologous regions, Box A and Box B. The Tob/BTG family proteins are involved in cell cycle regulation in a variety of cells such as T lymphocytes, fibroblasts, epithelial cells, and germ cells. In Tob-deficient mice, the incidence of liver tumors is higher than in wild-type mice. Furthermore, because the levels of tob expression are often repressed in human lung cancers, suppression of its expression is thought to contribute to tumor progression (13).The antiproliferative activities of the Tob/BTG family proteins are due to their association with target proteins in cells. For example, Tob associates with SMAD family proteins and acts as a negative regulator of SMAD signaling. In osteoblasts, this negative regulation occurs via association with SMAD 1, 5, 6, and 8 (14, 15), and via association with SMAD 2 and 4 in anergic quiescent T cells (16). Tob/BTG family proteins also bind to protein arginine methyltransferase, which regulates chromatin assembly by histone methylation (17). Much evidence has been accumulated to suggest that CCR4-associated factor 1 (Caf1),² also known as Cnot7 and involved in the CCR4-Not deadenylase complex, is a common binding partner of the Tob/BTG family proteins (4, 18-21). To reveal the functions of Caf1 in vivo, caf1^-/- mice have been generated in two groups. Male caf1-deficient mice are infertile because of a malfunction of the testicular somatic cells that leads to a defect in spermatogenesis (22, 23). Genetic analysis of the nematode C. elegans also suggests that FOG3 (Tob orthologue) interacts with CCF1, the C. elegans homologue of Caf1, and that this interaction is essential for germ cells to initiate spermatogenesis (24).Mouse and human Caf1 (mCaf1 and hCaf1) were found as homologues of yeast Pop2, a component of the CCR4-Not complex (18, 25). Yeast Pop2 displays weak RNase activity but enhances the deadenylation of the poly(A) tail of mRNA by the CCR4-Not deadenylase complex (26-29). The primary structure of mammalian Caf1 is related to that of the ribonuclease D (RNase D) family, and all of the active site residues are well conserved (30). Indeed, both mCaf1 and hCaf1 have deadenylase activity (31-33).Although the relationship between cell cycle repression and poly(A) deadenylation is not well understood, mRNA degradation and synthesis are major events in maintaining the cell cycle (34). The mRNAs in a eukaryotic cell have a wide range of half-lives. Degradation of mRNA is initiated by shortening of the poly(A) tail. Thereafter, the 5′-cap structure is removed and the remaining portion of the mRNA is rapidly degraded. The degradation of eukaryotic mRNAs is regulated precisely at each stage of the cell cycle. Tob was reported to associate with inducible poly(A)-binding protein (iPABP) and to abrogate the translation of interleukin-2 mRNA in vitro (35). Recent reports also showed that Tob and BTG2 interact with the CCR4-Not deadenylase complex using the Tob/BTG2 domain and the cytoplasmic poly(A)-binding protein (PABPC1) using the C-terminal tail and enhanced mRNA degradation (36-38).To help elucidate the relationship between the antiproliferative activity of Tob and the degradation of the poly(A) tail, we determined the crystal structure of the Tob-hCaf1 complex. We found that hCaf1 has a structure similar to yeast Pop2 and human PARN of deadenylases, exonuclease I, and the Klenow fragment of DNA polymerase I from Escherichia coli. In contrast, Tob has a novel structure. Specifically, Box A and Box B mediate the interaction between Tob and hCaf1. Cell growth assays using the wild and mutant proteins, together with the structural studies, revealed that the complex formation is crucial to cell growth inhibition.     

Detection of living <Emphasis Type="Italic">Salmonella</Emphasis> cells using bioluminescence

ATP-based bioluminescence using mutant firefly luciferase was combined with an immunochromatographic lateral flow test strip assay for Salmonella enteritidis detection. In this combination method, the Salmonella-antibody–gold complex captured at the test line on the test strip was lysed by heat-treatment, and the ATP released from the cells was measured using mutant luciferase. This method resulted in approximately 1,000 times higher sensitivity in the detection of Salmonella (i.e. 10³ c.f.u./ml) compared to immunochromatographic lateral flow assay.     

Murine double-minute 2 homolog single nucleotide polymorphism 309 and the risk of gynecologic cancer

A functional T to G germline polymorphism in the promoter region of murine double-minute 2 homolog single nucleotide polymorphism 309 (MDM2-SNP309) has been reported to profoundly accelerate tumor formation, suggesting that it may also represent a powerful cancer predisposing allele. In this study, MDM2-SNP309 was examined in a total of 400 blood samples from 108 normal, 88 cervical, 119 endometrial and 85 ovarian cancer cases using two independent polymerase chain reaction assays for each allele. When the MDM2-SNP309 genotype was classified into two subgroups of TT+TG and GG, the GG genotype was associated with an increased risk for the development of endometrial cancer (odds ratio [OR]= 1.91, 95% confidence interval [CI] = 1.05 to 3.47) compared with the TT+TG genotype ( P  = 0.0353). The G allele also increased the risk of endometrial cancer (OR = 1.20, 95% CI = 0.83 to 1.74) compared with the T allele, but no statistical difference was found ( P  = 0.3333). The homozygous GG genotype was also associated with postmenopausal status and type I endometrial cancer ( P  = 0.0306 and 0.0326, respectively). There was no significant difference in the genotype or allele prevalence between control subjects and cervical or ovarian cancer patients. These results suggest that homozygous GG genotype of MDM2-SNP309 may be a risk factor for postmenopausal and type I endometrial cancer in a Japanese population.