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PubMed2005

Expression of S100 proteins in normal human tissues and common cancers using tissue microarrays: S100A6, S100A8, S100A9 and S100A11 are all overexpressed in common cancers.

AIMS: To survey the expression of members of the S100 family of calcium-binding proteins in normal human tissues and common cancers using tissue microarrays. S100A6, S100A8, S100A9 and S100A11 have all been suggested to have potential roles in carcinogenesis and tumour progression but their expression has not been described in a wide range of human tissues and tumours. METHODS AND RESULTS: A custom-made tissue array, containing 291 tissue cores representing 28 tissue types and 21 tumour types, was used to produce sections that were immunostained for S100A2, S100A6, S100A8, S100A9, S100A11, calbindin 1, calbindin 2, S100B and parvalbumin. S100A6, S100A8 and S100A9 were expressed in 32%, 12% and 28% of breast cancers, respectively. There was a translocation of S100A11 expression from exclusively nuclear in normal tissues to cytoplasmic and nuclear in all common cancers. CONCLUSIONS: S100A6, S100A8, S100A9 and S100A11 are all expressed in common cancers, especially breast cancer. In addition, S100A11 undergoes a nucleocytoplasmic translocation which may have a direct influence on the proliferation of the cancer cells.

Calbindin 1↗
PubMed2001

Threading of a glycosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin subunits.

Chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone essential for human reproduction. Twenty hCG beta-subunit residues, termed the seatbelt, are wrapped around alpha-subunit loop 2 (alpha 2) and their positions "latched" by a disulfide formed by cysteines at the end of the seatbelt (Cys 110) and in the beta-subunit core (Cys 26). This unique arrangement explains the stability of the heterodimer but raises questions as to how the two subunits combine. The seatbelt is latched in the free beta-subunit. If the seatbelt remained latched during the process of subunit combination, formation of the heterodimer would require alpha 2 and its attached oligosaccharide to be threaded through a small beta-subunit hole. The subunits are known to combine during oxidizing conditions in vitro, and studies described here tested the idea that this requires transient disruption of the latch disulfide, possibly as a consequence of the thioredoxin activity reported in hCG. We observed that alkylating agents did not modify either cysteine in the latch disulfide (Cys 26 or Cys 110) during heterodimer formation in several oxidizing conditions and had minimal influence on these cysteines during combination in the presence of mild reductants (1--3 mM beta-mercaptoethanol). Reducing agents appeared to accelerate subunit combination by disrupting a disulfide (Cys 93--Cys 100) that forms a loop within the seatbelt, thereby increasing the size of the beta-subunit hole. We propose a mechanism for hCG assembly in vitro that depends on movements of alpha 2 and the seatbelt and suggest that the process of glycoprotein hormone subunit combination may be useful for studying the movements of loops during protein folding.

Alkylating Agents↗
PubMed2005

Cooperative binding of DNA and CBFbeta to the Runt domain of the CBFalpha studied via MD simulations.

The Runt domain (RD) is the DNA-binding region of the Runx genes. A related protein, known as core binding factor beta (CBFbeta) also binds to the RD to enhance RD-DNA interaction by 6- to 10-fold. Here, we report results from molecular dynamics (MD) simulations of RD alone, as a dimer in complexes with DNA and CBFbeta and in a ternary complex with DNA and CBFbeta. Consistent with the experimental findings, in the presence of CBFbeta the estimated free energy of binding of RD to the DNA is more favorable, which is shown to be due to more favorable intermolecular interactions and desolvation contributions. Also contributing to the enhanced binding are favorable intramolecular interactions between the 'wing' residues (RD residues 139-145) and the 'wing1' residues (RD residues 104-116). The simulation studies also indicate that the RD-CBFbeta binding is more favorable in the presence of DNA due to a more favorable RD-CBFbeta interaction energy. In addition, it is predicted that long-range interactions involving ionic residues contribute to binding cooperativity. Results from the MD calculations are used to interpret a variety of experimental mutagenesis data. A novel role for RD Glu116 to the RD-CBFbeta interaction is predicted.

Amino Acids↗
PubMed1997

Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166-171) and the Mg2+ positioning loop (residues 184-186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg x ATP and the peptide inhibitor. In this ternary complex the gamma-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the gamma-subsite.

Adenine↗
PubMed2004

Familial mutations of the transcription factor RUNX1 (AML1, CBFA2) predispose to acute myeloid leukemia.

RUNX1 (AML1, CBFA2) is mutated in affected members of families with autosomal dominant thrombocytopenia and platelet dense granule storage pool deficiency. Many of those affected, usually by point mutations in one allele, are predisposed to the development of acute myeloid leukemia (AML) in adult life. The RUNX1 protein complexes with core binding factor beta (CBFB) to form a heterodimeric core binding transcription factor (CBF) that regulates many genes important in hematopoiesis. RUNX1 was first identified as the gene on chromosome 21 that is rearranged by the translocation t(8;21)(q22;q22.12) recurrently found in the leukemic cells of patients with AML. In addition to the t(8;21), RUNX1 is rearranged with one of several partner genes on other chromosomes by somatically acquired translocations associated with hematological malignancies. Point mutations of RUNX1 are also found in sporadic leukemias to reinforce the important position of this gene on the multi-step path to leukemia. In animal models, at least one functional copy of RUNX1 is required to effect definitive embryonic hematopoiesis. Cells expressing dominant-negative mutants of RUNX1 are readily immortalized and transformed, and those RUNX1 mutants which retain CBFB binding ability may possess dominant-negative function. However, in some families there is transmitted one mutated allele of RUNX1 with no dominant-negative function, demonstrating that simple haploinsufficiency of RUNX1 predisposes to AML and also causes a generalized hematopoietic stem cell disorder most recognizable as thrombocytopenia.

Animals↗
PubMed2006

Deletion of the rpoZ gene, encoding the omega subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis.

The omega subunit, the smallest subunit of bacterial RNA polymerase, is known to be involved in maintaining the conformation of the beta' subunit and aiding its recruitment to the rest of the core enzyme assembly in Escherichia coli. It has recently been shown in Mycobacterium smegmatis, by creating a deletion mutation of the rpoZ gene encoding omega, that the physiological role of the omega subunit also includes providing physical protection to beta'. Interestingly, the mutant had altered colony morphology. This paper demonstrates that the mutant mycobacterium has pleiotropic phenotypes including reduced sliding motility and defective biofilm formation. Analysis of the spatial arrangement of biofilms by electron microscopy suggests that the altered phenotype of the mutant arises from a deficiency in generation of extracellular matrix. Complementation of the mutant strain with a copy of the wild-type rpoZ gene integrated in the bacterial chromosome restored both sliding motility and biofilm formation to the wild-type state, unequivocally proving the role of omega in the characteristics observed for the mutant bacterium. Analysis of the cell wall composition demonstrated that the mutant bacterium had an identical glycopeptidolipid profile to the wild-type, but failed to synthesize the short-chain mycolic acids characteristic of biofilm growth in M. smegmatis.

Bacterial Proteins↗
PubMed1996

The chimeric genes AML1/MDS1 and AML1/EAP inhibit AML1B activation at the CSF1R promoter, but only AML1/MDS1 has tumor-promoter properties.

The (3;21)(q26;q22) translocation associated with treatment-related myelodysplastic syndrome, treatment-related acute myeloid leukemia, and blast crisis of chronic myeloid leukemia results in the expression of the chimeric genes AML1/EAP, AML1/MDS1, and AML1/EVI1. AML1 (CBFA2), which codes for the alpha subunit of the heterodimeric transcription factor CBF, is also involved in the t(8;21), and the gene coding for the beta subunit (CBFB) is involved in the inv(16). These are two of the most common recurring chromosomal rearrangements in acute myeloid leukemia. CBF corresponds to the murine Pebp2 factor, and CBF binding sites are found in a number of eukaryotic and viral enhancers and promoters. We studied the effects of AML1/EAP and AML1/MDS1 at the AML1 binding site of the CSF1R (macrophage-colony-stimulating factor receptor gene) promoter by using reporter gene assays, and we analyzed the consequences of the expression of both chimeric proteins in an embryonic rat fibroblast cell line (Rat1A) in culture and after injection into athymic nude mice. Unlike AML1, which is an activator of the CSF1R promoter, the chimeric proteins did not transactivate the CSF1R promoter site but acted as inhibitors of AML1 (CBFA2). AML1/EAP and AML1/MDS1 expressed in adherent Rat1A cells decreased contact inhibition of growth, and expression of AML1/MDS1 was associated with acquisition of the ability to grow in suspension culture. Expression of AML1/MDS1 increased the tumorigenicity of Rat1A cells injected into athymic nude mice, whereas AML1/EAP expression prevented tumor growth. These results suggest that expression of AML1/EAP and AML1/MDS1 can interfere with normal AML1 function, and that AML1/MDS1 has tumor-promoting properties in an embryonic rat fibroblast cell line.

Acute Disease↗
PubMed1999

HERF1, a novel hematopoiesis-specific RING finger protein, is required for terminal differentiation of erythroid cells.

The AML1/core binding factor beta (CBFbeta) transcription factor is essential for definitive hematopoiesis; however, the downstream pathways through which it functions remain incompletely defined. Using a differential cloning approach to define components of this pathway, we have identified a novel gene designated HERF1 (for hematopoietic RING finger 1), whose expression during development is dependent on the presence of functional AML1/CBFbeta. HERF1 contains a tripartite RING finger-B box-alpha-helical coiled-coil domain and a C-terminal region homologous to the ret proto-oncogene-encoded finger protein. Expression of HERF1 during embryogenesis coincides with the appearance of definitive erythropoiesis and in adult mice is restricted to erythroid cells, increasing 30-fold during terminal differentiation. Importantly, inhibition of HERF1 expression blocked terminal erythroid differentiation of the murine erythroleukemia cell line MEL, whereas its overexpression induced erythroid maturation. These results suggest an important role for this protein in erythropoiesis.

Animals↗
PubMed2005

Functional characterization of core promoter elements: the downstream core element is recognized by TAF1.

Downstream elements are a newly appreciated class of core promoter elements of RNA polymerase II-transcribed genes. The downstream core element (DCE) was discovered in the human beta-globin promoter, and its sequence composition is distinct from that of the downstream promoter element (DPE). We show here that the DCE is a bona fide core promoter element present in a large number of promoters and with high incidence in promoters containing a TATA motif. Database analysis indicates that the DCE is found in diverse promoters, supporting its functional relevance in a variety of promoter contexts. The DCE consists of three subelements, and DCE function is recapitulated in a TFIID-dependent manner. Subelement 3 can function independently of the other two and shows a TFIID requirement as well. UV photo-cross-linking results demonstrate that TAF1/TAF(II)250 interacts with the DCE subelement DNA in a sequence-dependent manner. These data show that downstream elements consist of at least two types, those of the DPE class and those of the DCE class; they function via different DNA sequences and interact with different transcription activation factors. Finally, these data argue that TFIID is, in fact, a core promoter recognition complex.

Adenoviridae↗
PubMed1994

Decreased ternary complex formation and predominance of a 29 kDa IGFBP-3 fragment in human fetal serum.

Insulin-like growth factor-I (IGF-I) has been proposed to be important in the endocrine control of fetal growth in humans, although serum IGF-I concentrations are 10-fold lower than during rapid pubertal growth. However, the bioavailability of IGF-I in fetal serum may be increased by changes in the specific IGF binding proteins (IGFBPs). We have recently suggested that the bioavailability of circulating IGF-I is increased in the human fetus due to the molar excess of IGF-I plus IGF-II relative to IGFBP-3 as well as the increased concentrations of IGFBP-2, which does not form a long-lived ternary complex. We have presently studied ternary complex formation between IGF, IGFBP-3, and acid labile subunit (ALS) to further assess if IGF-I bioavailability is increased in human fetal serum. In 19-35 week gestation fetal sera, a markedly decreased formation of the ternary complex was demonstrated by the general absence of IGFBP-3 (detected by Western immunoblotting) in the approximately 130-150 kDa ternary complex after neutral size chromatography. The predominant form of IGFBP-3 in fetal serum was a 29 kDa fragment, which, following deglycosylation by Endoglycosidase-F, was demonstrated to consist of a approximately 20 kDa protein core. Despite the predominance of the 29 kDa IGFBP-3 fragment, we have previously demonstrated that the IGFBP-3 protease activity is not increased in fetal serum, in contrast to pregnancy or non-insulin dependent diabetes mellitus (NIDDM) sera.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2005

Alveolar bone osteoblast differentiation and Runx2/Cbfa1 expression.

Alveolar bone cells have a unique origin and functionality, but may resemble skeletal osteoblasts. Osteoblast differentiation and gene expression are regulated by the Runx2/Cbfa1 transcription factor. However, most studies on Runx2/Cbfa1 expression have been on rodent cells and the few studies on human osteoblasts have had differing results. The purpose of this study was to characterize Runx2/Cbfa1 expression in primary cell cultures derived from human alveolar bone. An alveolar bone chip was incubated in alpha-minimum essential medium (alpha-MEM) supplemented with fetal calf serum (10% FCS). Explant cultures were harvested after 3-4 weeks of outgrowth and grown in alpha-MEM with FCS. This media was supplemented with ascorbate, beta-glycerophosphate and dexamethasone to promote osteoblast differentiation over 14 days. RT-PCR analysis and Western blots showed a rapid increase in Runx2/Cbfa1 mRNA (2.1-fold) and protein (2.3-fold) levels in 3 days, followed by a slight decline. There was also a rapid increase in bone sialoprotein expression (2.9-fold) in 3 days, followed by a further increase (3.6-fold) at 14 days. There was a slower increase in alkaline phosphatase expression (1.6-fold) and activity (3.1-fold) over 7 days, followed by a gradual decline. In contrast, collagen mRNA levels showed little change over 14 days. These findings attest to the osteogenic potential of primary cell cultures derived from human alveolar bone. Osteoblastic differentiation in human alveolar bone involves an increase in Runx2/Cbfa1 expression that may be an important component of the differentiation process.

Alkaline Phosphatase↗
PubMed1993

Synthetic human beta-globin 5'HS2 constructs function as locus control regions only in multicopy transgene concatamers.

Transgenes linked to the beta-globin locus control region (LCR) are transcribed in a copy-dependent manner that is independent of the integration site. It has previously been shown that the LCR 5'HS2 region does not require its NF-E2 dimer binding site for LCR activity. In this paper we analyse synthetic 5'HS2 core constructs containing point mutations in the other factor binding sites 3' of the NF-E2 dimer site. The results show that 5'HS2 core is a partially active LCR that functions in a concatamer of at least two copies but not when present as a single copy in transgenic mice and that no single binding site within 5'HS2 is required for position-independent expression. In addition, the H-BP factor is identical to upstream stimulatory factor (USF) and full enhancement levels by 5'HS2 core in MEL cells require a combination of all the factor binding sites. We suggest that 5'HS2 cores in a concatamer interact with each other to establish an area of open chromatin and that this process may be the basis of LCR function.

Animals↗
PubMed1999

Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPARgamma2.

Cells of the bone marrow stroma can reversibly convert among different phenotypes. Based on this and on evidence for a reciprocal relationship between osteoblastogenesis and adipogenesis, we have isolated several murine bone marrow-derived clonal cell lines with phenotypic characteristics of osteoblasts or adipocytes, or both. Consistent with a state of plasticity, cell lines with a mixed phenotype synthesized osteoblast markers like type I collagen, alkaline phosphatase, osteocalcin, as well as the adipocyte marker lipoprotein lipase, under basal conditions. In the presence of ascorbic acid and beta-glycerophosphate-agents that promote osteoblast differentiation-they formed a mineralized matrix. In the presence of isobutylmethylxanthine, hydrocortisone, and indomethacin-agents that promote adipocyte differentiation-they accumulated fat droplets, but failed to express adipsin and aP2, markers of terminally differentiated adipocytes. Furthermore, they were converted back to matrix mineralizing cells when the adipogenic stimuli were replaced with the osteoblastogenic ones. A prototypic cell line with mixed phenotype (UAMS-33) expressed Osf2/Cbfa1-a transcription factor required for osteoblast differentiation, but not PPARgamma2-a transcription factor required for terminal adipocyte differentiation. Stable transfection with a PPARgamma2 expression construct and activation with the thiazolidinedione BRL49653 stimulated aP2 and adipsin synthesis and fat accumulation, and simultaneously suppressed Osf2/Cbfa1, alpha1(I) procollagen, and osteocalcin synthesis. Moreover, it rendered the cells incapable of forming a mineralized matrix. These results strongly suggest that PPARgamma2 negatively regulates stromal cell plasticity by suppressing Osf2/Cbfa1 and osteoblast-like biosynthetic activity, while promoting terminal differentiation to adipocytes.

Adipocytes↗
PubMed2005

Effects of mechanical strain on proliferation and differentiation of bone marrow stromal cell line ST2.

Differentiation of mesenchymal stromal cells into osteoblasts is regulated by many factors including growth factors, cytokines, and hormones. Mechanical stress has been considered to be an important factor in bone modeling and remodeling. However, biological responses of stromal cells to mechanical stimuli are still unknown. To show the correlation between magnitude of mechanical strain and differentiation of stromal cells into osteoblasts, we investigated the proliferation and the expression of osteoblast-related genes in stromal cell line ST2 that is in the process of osteoblastic differentiation by treatment with ascorbic acid and beta-glycerophosphate, under 0.8%-15% elongation using the Flexercell Strain system. The expression of osteoblast-related genes was analyzed by real-time quantitative polymerase chain reaction (PCR). Cell proliferation significantly increased at 5%, 10%, and 15% elongation compared to that of unloaded controls. Alkaline phosphatase (ALPase) activity significantly increased at 0.8% and 5% elongation but decreased at 10% and 15% elongation. At 1 h and 6 h, mRNA level of Cbfa1/Runx2 increased at lower magnitudes of strain (0.8% and 5% elongation) but decreased at higher magnitude of strain (15% elongation). At 24 and 48 h, Cbfa1/Runx2 and osteocalcin mRNAs decreased at 5%, 10%, and 15% elongation, whereas cell proliferation and expression of type I collagen mRNA increased at the same elongation. These results indicate that mechanical strain stimulates osteoblastic differentiation of stromal cells at low magnitudes of strain.

Alkaline Phosphatase↗
PubMed2004

Analysis of beta-1,3-glucan assembly in Saccharomyces cerevisiae using a synthetic interaction network and altered sensitivity to caspofungin.

Large-scale screening of genetic and chemical-genetic interactions was used to examine the assembly and regulation of beta-1,3-glucan in Saccharomyces cerevisiae. Using the set of deletion mutants in approximately 4600 nonessential genes, we scored synthetic interactions with genes encoding subunits of the beta-1,3-glucan synthase (FKS1, FKS2), the glucan synthesis regulator (SMI1/KNR4), and a beta-1,3-glucanosyltransferase (GAS1). In the resulting network, FKS1, FKS2, GAS1, and SMI1 are connected to 135 genes in 195 interactions, with 26 of these genes also interacting with CHS3 encoding chitin synthase III. A network core of 51 genes is multiply connected with 112 interactions. Thirty-two of these core genes are known to be involved in cell wall assembly and polarized growth, and 8 genes of unknown function are candidates for involvement in these processes. In parallel, we screened the yeast deletion mutant collection for altered sensitivity to the glucan synthase inhibitor, caspofungin. Deletions in 52 genes led to caspofungin hypersensitivity and those in 39 genes to resistance. Integration of the glucan interaction network with the caspofungin data indicates an overlapping set of genes involved in FKS2 regulation, compensatory chitin synthesis, protein mannosylation, and the PKC1-dependent cell integrity pathway.

Antifungal Agents↗
PubMed2004

Normal-mode analysis suggests protein flexibility modulation throughout RNA polymerase's functional cycle.

To explore the domain-scale flexibility of bacterial RNA polymerase (RNAP) throughout its functional cycle, block normal-mode analyses (BNM) were performed on several important functional states, including the holoenzyme, the core complex, a model of RNAP bound to primarily duplex DNA, and a model of the ternary elongation complex. The calculations utilized a molecular mechanics (MM) force field with physical interactions; this is made possible by the use of BNM and the implementation of a sparse-matrix diagonalization routine. The use of homology models necessitated the MM force field rather than the simpler elastic network model (ENM). From the MM/BNM, we have systematically and semiquantitatively calculated the atomic fluctuations in the four functional states without bias due to crystal packing or other artifactual forces. We have observed that both alpha subunits and the omega subunit are rigid, in line with their roles as structural motifs that are not mechanistically involved in RNAP's functional cycle. It has been observed that the beta subunit has two highly mobile domains; these are commonly known as the beta1 and beta2 domains. Our calculations suggest that the flexibility of these domains is modulated throughout the functional cycle and that they move entirely independently of each other unless DNA is bound. From an energetic perspective, we have shown the beta2 domain can flex into and out of the cleft, forming interactions with DNA in the TEC as has been previously proposed. Our calculations also confirm that the beta' subunit's likely flexibility into and out of the DNA binding cleft is energetically allowed. These two observations validate that both of the RNAP crab claw's pincers are mobile, as both beta and beta' have substantial flexibility.

Bacterial Proteins↗
PubMed2000

The SCF(HOS/beta-TRCP)-ROC1 E3 ubiquitin ligase utilizes two distinct domains within CUL1 for substrate targeting and ubiquitin ligation.

We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IkappaBalpha. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCF(HOS/beta-TRCP)-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/beta-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IkappaB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2006

[Effects of core binding factor alpha1 on promotion of osteoblastic differentiation from marrow mesenchymal stem cells].

OBJECTIVE: To observe effects of the core binding factor alpha1 (Cbfalpha1) in its promoting differentiation of the rabbit marrow mesenchymal stem cells (MSCs) into osteoblasts. METHODS: The rabbit marrow MSCs were isolated and cultured in vitro and were divided into 3 groups. In the control group, the marrow MSCs were cultured by DMEM; in the single inducement group, they were cultured by the condition medium (DMEM, 10% fetal bovine serum, dexamethasone 10 mmol/L, vitamin C 50 mg/L, and beta-GP 10 mmol/L); and in the experimental group, they were transfected with AdEasyl/Cbfalpha1, and then were cultured by the condition medium. The alkaline phosphatase (ALP) activity and the expression of osteocalcin as the osteoblast markers were measured with the chemohistological and immunohistochemical methods at 3 days, 1, 2, 3, and 4 weeks after inducement. RESULTS: More than 90% MSCs were grown well in vitro. The GFP was positive in MSCs after their being transfected with AdEasyl/Cbfalpha1. The ALP activity and the expression of osteocalcin were significantly upregulated in the transfection group compared with those in the single inducement group and the control group at 1, 2, 3, and 4 weeks (P < 0.05). The mineralized node began to appear at 2 weeks in the experimental group and the single induction group, but did not appear in control group. CONCLUSION: Cbfalpha1 can obviously promote differentiation of the rabbit marrow mesenchymal stem cells into the osteoblasts.

Animals↗