Structural basis for regulation of the Crk signaling protein by a proline switch

Proline switches, controlled by cis-trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. Here we report the structures of both the cis and trans conformers of a proline switch in the Crk signaling protein. Proline isomerization toggles Crk between two conformations: an autoinhibitory conformation, stabilized by the intramolecular association of two tandem SH3 domains in the cis form, and an uninhibited, activated conformation promoted by the trans form. In addition to acting as a structural switch, the heterogeneous proline recruits cyclophilin A, which accelerates the interconversion rate between the isomers, thereby regulating the kinetics of Crk activation. The data provide atomic insight into the mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency. The results also reveal new SH3 binding surfaces, highlighting the binding versatility and expanding the noncanonical ligand repertoire of this important signaling domain.     

Apelin promotes osteosarcoma metastasis by upregulating PLOD2 expression via the Hippo signaling pathway and hsa_circ_0000004/miR-1303 axis

Osteosarcoma is a highly mortal bone tumor, with a high metastatic potential, promoted in part by the enzyme procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2). Increasing level of PLOD2 in osteosarcoma tissue correlates with lymphatic and distant metastasis. The adipokine apelin (APLN) is also found in different cancers and APLN upregulation promotes angiogenesis and metastasis, but its effects on osteosarcoma metastasis are uncertain. We explored APLN functioning in metastatic osteosarcoma. An analysis of records from the Gene Expression Omnibus (GEO) database showed higher levels of APLN expression in osteosarcoma tissue than in normal tissue. Similarly, levels of APLN and PLOD2 mRNA synthesis were upregulated in osteosarcoma tissue. Levels of APLN and PLOD2 protein correlated positively with osteosarcoma clinical stages. APLN increased PLOD2 expression in human osteosarcoma cell lines and cell migration via the mammalian Sterile 20-like kinase 1 (MST1), monopolar spindle-one-binder protein (MOB)1, and YAP cascades, and through hsa_circ_0000004 functioning as a sponge of miR-1303. We also found that knockdown of APLN antagonized lung metastasis in mice with osteosarcoma. APLN may be a therapeutic target in osteosarcoma metastasis.     

Effects of cis and trans regulatory variations on the expression divergence of heat shock response genes between yeast strains

Phenotypic variation among individuals in a population can be due to DNA sequence variation in protein coding regions or in regulatory elements. Recently, many studies have indicated that mutations in regulatory elements may be the major cause of phenotypic evolution. However, the mechanisms for evolutionary changes in gene expression are still not well understood. Here, we studied the relative roles of cis and trans regulatory changes in Saccharomyces cerevisiae cells to cope with heat stress. It has been found that the expression level of ~ 300 genes was induced at least two fold and that of ~ 500 genes was repressed at least two fold in response to heat shock. From the former set of genes, we randomly selected 65 genes that showed polymorphism(s) between the BY and RM strains for pyrosequencing analysis to explore the relative contributions of cis and trans regulatory variations to the expression divergence between BY and RM. Our data indicated that the expression divergence between BY and RM was mainly due to trans regulatory variations under either the normal condition or the heat stress condition. However, the relative contribution of trans regulatory variation was decreased from 76.9% to 61.5% after the heat shock stress. These results indicated that the cis regulatory variation may play an important role in the adaption to heat stress. In our data, 43.1% (28 genes) of the 65 genes showed the same trend of cis or trans variation effect after the heat shock stress, 35.4% (23 genes) showed an increased cis variation effect and 21.5% (14 genes) showed an increased trans variation effect after the heat shock stress. Thus, our data give insights into the relative roles of cis and trans variations in response to heat shock in yeast.     

Synthesis, antiproliferative, and vasorelaxing evaluations of coumarin alpha-methylene-gamma-butyrolactones

Certain coumarin alpha-methylene-gamma-butyrolactones were synthesized and evaluated for antiproliferative and vasorelaxing activities. These compounds were synthesized via alkylation of hydroxycoumarins 2a-f followed by oxidation and the Reformatsky-type condensation. The results of this study are as follows (1) for the vasorelaxing activity, coumarin-7-yl alpha-methylene-gamma-butyrolactone 6d, with an IC50 value of 9.4 microM against pig coronary arterial contraction induced by KCl, is a more active vasorelaxant than its coumarin-4-yl counterpart 6a and its gamma-methyl congener 1. A methyl group substituted at C-4 of the coumarin-7-yl moiety reduced the vasorelaxing effect (6d vs 6e) while the 3,4,8-trimethyl derivative 6f was inactive. (2) For the antiproliferative activity, coumarin-4-yl alpha-methylene-gamma-butyrolactone 6a, which exhibited the most potent antiproliferative activity on the growth of MCF7, NCI-H460, and SF-268 with IC50 values of 6.97, 14.68, and 8.36 microM, respectively, is more cytotoxic than its coumarin-7-yl counterpart 6d and the 6,7-dimethyl derivative 6b. For the coumarin-7-yl derivatives, 6d is more active than its gamma-methyl congener 1, indicating that substitution at the gamma-position decreased cytotoxicity.     

Chloramphenicol-induced mitochondrial stress increases p21 expression and prevents cell apoptosis through a p21-dependent pathway

Pretreatment of HepG2 and H1299 cells with chloramphenicol rendered the cells resistant to mitomycin-induced apoptosis. Both mitomycin-induced caspase 3 activity and PARP activation were also inhibited. The mitochondrial DNA-encoded Cox I protein, but not nuclear-encoded proteins, was down-regulated in chloramphenicol-treated cells. Cellular levels of the p21(waf1/cip1) protein and p21(waf1/cip1) mRNA were increased through a p53-independent pathway, possibly because of the stabilization of p21(waf1/cip1) mRNA in chloramphenicol-treated cells. The p21(waf1/cip1) was redistributed from the perinuclear region to the cytoplasm and co-localized with mitochondrial marker protein. Several morphological changes and activation of the senescence-associated biomarker, SA beta-galactosidase, were observed in these cells. Both p21(waf1/cip1) antisense and small interfering RNA could restore apoptotic-associated caspase 3 activity, PARP activation, and sensitivity to mitomycin-induced apoptosis. Similar effects were seen with other antibiotics that inhibit mitochondrial translation, including minocycline, doxycycline, and clindamycin. These findings suggested that mitochondrial stress causes resistance to apoptosis through a p21-dependent pathway.     

The B cell inhibitory Fc receptor triggers apoptosis by a novel c-Abl family kinase-dependent pathway

The inhibitory Fc receptors function to regulate the antigen-driven activation and expansion of lymphocytes. In B cells, the Fc gammaRIIB1 is a potent inhibitor of B cell antigen receptor (BCR) signaling when coligated to the BCR by engagement of antigen-containing immune complexes. Inhibition is mediated by the recruitment of the inositol phosphatase, SHIP, to the Fc gammaRIIB1 phosphorylated tyrosine-based inhibitory motif (ITIM). Here we show that BCR-independent aggregation of the Fc gammaRIIB1 transduces an ITIM- and SHIP-independent proapoptotic signal that is dependent on members of the c-Abl tyrosine kinase family. These results define a novel Abl family kinase-dependent Fc gammaRIIB1 signaling pathway that functions independently of the BCR in controlling antigen-driven B cell responses.     

Photophysical properties, electronic and crystal structures of luminescent diphosphine digold(I)-pyridine-2-thiolate complexes

Treatment of diphosphine digold(I) complexes, PP(AuCl)₂ [PP=bis(diphenylphosphino)methane, dppm; 1,6-bis(diphenylphosphino)hexane, dpph], with two equivalents of pyridine-2-thiol (HNS) in the presence of NaOCH₃ affords two luminescent diphosphine digold(I)-pyridine-2-thiolate complexes, dppm(AuSN)₂ (1) and dpph(AuSN)₂ (2), respectively. Both crystal structures have been determined by crystallographic studies. The intramolecular aurophilic contact of 3.0478(3) Å is observed in the crystal structure of 1, whereas there is not any aurophilic interaction present in the crystal lattices of 2. At room temperature, 1 shows a low-energy emission at ca. 660 nm as well as a very weak high-energy emission at ca. 496 nm in the solid state, but 2 shows only a strong high-energy one at ca. 482 nm. Thus, the emission energy strongly dependent on the Au(I)?Au(I) interaction can be demonstrated in the diphosphine digold(I) thiolates studied herein.     

The C-terminal sequence of LMADS1 is essential for the formation of homodimers for B function proteins

LMADS1, a lily (Lilium longiflorum) AP3 orthologue, contains the complete consensus sequence of the paleoAP3 (YGSHDLRLA) and PI-derived (YEFRVQPSQPNLH) motifs in the C-terminal region of the protein. Interestingly, through yeast two-hybrid analysis, LMADS1 was found to be capable of forming homodimers. These results indicated that LMADS1 represents an ancestral form of the B function protein, which retains the ability to form homodimers in regulating petal and stamen development in lily. To explore the involvement of the conserved motifs in the C-terminal region of LMADS1 in forming homodimers, truncated forms of LMADS1 were generated, and their ability to form homodimers was analyzed using yeast two-hybrid and electrophoretic mobility shift assay. The ability of LMADS1 to form homodimers decreased once the C-terminal paleoAP3 motif was deleted. When both paleoAP3 and PI-derived motifs were deleted, the ability of LMADS1 to form homodimers was completely abolished. This result indicated that although the paleoAP3 motif promotes the formation of LMADS1 homodimers, the PI-derived motif is essential. Deletion analysis indicated that two amino acids, RV, of the 5 final amino acids, YEFRV, in the PI-derived motif are essential for the formation of homodimers. Further, point mutation analysis indicated that amino acid Val was absolutely necessary, whereas residue Arg played a less important role in the formation of homodimers. Furthermore, Arabidopsis AP3 was able to form homodimers once its C-terminal region was replaced by that of LMADS1. This result indicated that the C-terminal region of LMADS1 is responsible and essential for homodimer formation of the ancestral form of the B function protein.     

Synthesis and antiproliferative evaluations of certain 2-phenylvinylquinoline (2-styrylquinoline) and 2-furanylvinylquinoline derivatives

The present study describes the synthesis of 2-phenylvinylquinoline (styrylquinoline) and 2-furanylvinylquinoline derivatives and evaluation for their antiproliferative activities. (E)-2-Styrylquinolin-8-ol (14a) was inactive against a 3-cell line panel consisting of MCF-7 (Breast), NCI-H460 (Lung), and SF-268 (CNS). Replacement of the phenyl ring with 5-nitrofuran-2-yl group significantly enhanced antiproliferative activity in which (E)-2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-8-ol (14i) and its 4-substituted derivatives 15–19 exhibited strong inhibitory effects against the growth of all three cancer cells. These compounds were further evaluated for their IC₅₀ against the growth of MCF-7, LNCaP, and PC3. Results indicated that a hydrogen bond donating oxime derivative 19a was more active than its hydrogen bond accepting methyloxime derivative 19b. For the inhibition of LNCaP, the potency decreased in an order 14i > 19a > 19b > 15 > 18 > 16. Compound 14i is the most active with an IC₅₀ value of 0.35 and 0.14 μM, respectively, against the growth of LNCaP and PC3 cancer cells. Therefore, compound 14i was evaluated by flow cytometric analysis for its effects on cell cycle distributions. Results indicated that 14i effectively induced cell cycle arrest at S phase for both cell lines, which consequently trigger late apoptosis for both LNCaP and PC3 cells.