Phospholipase Cgamma binds alpha1beta1 integrin and modulates alpha1beta1 integrin-specific adhesion.

Integrin adhesion receptors have been implicated in bidirectional signal transduction. The dynamic regulation of integrin affinity and avidity as well as post-ligand effects involved in outside-in signaling depends on the interaction of integrins with cytoskeletal and signaling proteins. In this study, we attempted to identify cytoplasmic binding partners of alpha(1)beta(1) integrin. We were able to show that cell adhesion to alpha(1)beta(1)-specific substrates results in the association of phospholipase Cgamma (PLCgamma) with the alpha(1)beta(1) integrin independent of PLCgamma tyrosine phosphorylation. Using peptide-binding assays, the membrane proximal sequences within the alpha(1)beta(1) integrin subunits were identified as binding sites for PLCgamma. In particular, the conserved sequence of beta(1) subunit binds the enzyme very efficiently. Because purified PLCgamma also binds the integrin peptides, binding seems to be direct. Inhibition of PLC by leads to reduced cell adhesion on alpha(1)beta(1)-specific substrates. Cells lacking the conserved domain of the alpha(1) subunit fail to respond to the PLC inhibition, indicating that this domain is necessary for PLC-dependent adhesion modulation of alpha(1)beta(1) integrin.     

The sphingosine-1-phosphate receptors S1P1, S1P2, and S1P3 function coordinately during embryonic angiogenesis

Sphingosine-1-phosphate (S1P) elicits diverse cellular responses through a family of G-protein-coupled receptors. We have shown previously that genetic disruption of the S1P(1) receptor, the most widely expressed of the family, results in embryonic lethality because of its key role within endothelial cells in regulating the coverage of blood vessels by vascular smooth muscle cells. To understand the physiologic functions of the two other widely expressed S1P receptors, we generated S1P(2) and S1P(3) null mice. Neither the S1P(2) null mice nor the S1P(3) null mice exhibited significant embryonic lethality or obvious phenotypic abnormalities. To unmask possible overlapping or collaborative functions between the S1P(1), S1P(2), and S1P(3) receptors, we examined embryos with multiple S1P receptor mutations. We found that S1P(1) S1P(2) double null and S1P(1) S1P(2) S1P(3) triple null embryos displayed a substantially more severe vascular phenotype than did embryos with only S1P(1) deleted. We also found partial embryonic lethality and vascular abnormalities in S1P(2) S1P(3) double null embryos. Our results indicate that the S1P(1), S1P(2) and S1P(3) receptors have redundant or cooperative functions for the development of a stable and mature vascular system during embryonic development.     

Inhibition of APCCdh1 activity by Cdh1/Acm1/Bmh1 ternary complex formation

The anaphase-promoting complex (APC) is an essential E3 ubiquitin ligase responsible for catalyzing proteolysis of key regulatory proteins in the cell cycle. Cdh1 is a co-activator of the APC aiding in the onset and maintenance of G(1) phase, whereas phosphorylation of Cdh1 at the end of G(1) phase by cyclin-dependent kinases assists in the inactivation of APC(Cdh1). Here, we suggest additional components are involved in the inactivation of APC(Cdh1) independent of Cdh1 phosphorylation. We have identified proteins known as Acm1 and Bmh1, which bind and form a ternary complex with Cdh1. The presence of phosphorylated Acm1 is critical for the ternary complex formation, and Acm1 is predominantly expressed in S phase when APC(Cdh1) is inactive. The assembly of the ternary complex inhibits ubiquitination of Clb2 in vitro by blocking the interaction of Cdh1 with Clb2. In vivo, lethality caused by overexpression of constitutively active Cdh1 is rescued by overexpression of Acm1. Partially phosphorylated Cdh1 in the absence of ACM1 still binds to and activates the APC. However, the addition of Acm1 decreases Clb2 ubiquitination when using either phosphorylated or nonphosphorylated Cdh1. Taken together, our results suggest an additional inactivation mechanism exists for APC(Cdh1) that is independent of Cdh1 phosphorylation.     

Arrhythmogenic action of endothelin-1(1-31) through conversion to endothelin-1(1-21)

Endothelin (ET)-1(1-21) is known to play an important role in the pathogenesis of acute ischemic arrhythmia. In the present study, we attempted to determine whether administration of ET-1(1-31) would result in arrhythmia in perfused isolated rat hearts. Forty-eight Sprague-Dawley rats weighing approximately 250-350 g were randomized into 6 groups. Heart was isolated and perfused in a Langendorff mode. The effects of ET-1(1-31) on arrhythmia, heart rate, coronary flow, and heart function were analyzed. Perfusion with 1 nM ET-1(1-31) resulted in frequent ventricular ectopic beats (VEBs) and ventricular tachycardia (VT). Overall VEB was 128.0 (approximately 66.0-1015.0), and the arrhythmia score (AS) was 2.18 +/- 0.87; both were significantly higher than those of the control group (P < 0.01). Pretreatment with perfusion of 10 nM of the ETA-receptor antagonist BQ(123) markedly attenuated the occurrence of VEB and VT induced by ET-1(1-31). AS in 10 nM BQ123 group was significantly lower than that in 1 nM ET-1(1-31) group (P < 0.01). The arrhythmia induced by 1 nM ET-1(1-31) was partially but significantly reduced by phosphoramidon (1 microM), a neutral endopeptidase/ET-converting enzyme inhibitor. ET-1(1-31) per se caused arrhythmia in perfused isolated rat hearts. This arrhythmogenic action is in part mediated by ET(A) receptor and may be attributed mainly to the conversion of ET-1(1-31) to ET-1(1-21.).     

Translation elongation factor-1A1 (eEF1A1) localizes to the spine by domain III

In vertebrates, there are two variants of eukaryotic peptide elongation factor 1A (eEF1A; formerly eEF-1α), eEF1A1 and eEF1A2, which have three well-conserved domains (D(I), D(II), and D(III)). In neurons, eEF1A1 is the embryonic type, which is expressed during embryonic development as well as the first two postnatal weeks. In the present study, EGFP-tagged eEF1A1 truncates were expressed in cortical neurons isolated from rat embryo (E18-19). Live cell images of transfected neurons showed that D(III)-containing EGFP-fusion proteins (EGFP-D(III), -D(II)-III, -D(I)-III) formed clusters that were confined within somatodendritic domains, while D(III)-missing ones (EGFP-D(I), -D(II), -D(I)-II) and control EGFP were homogeneously D(I)spersed throughout the neuron incluD(I)ng axons. In dendrites, EGFP-D(III) was targeted to the heads of spine- and filopoD(I)a-like protrusions, where it was colocalized with SynGAPα, a postsynaptic marker. Our data inD(I)cate that D(III) of eEF1A1 meD(I)ates formation of clusters and localization to spines.     

Lentiviral siRNA silencing of sphingosine-1-phosphate receptors S1P1 and S1P2 in smooth muscle

Sphingosine-1-phosphate regulates diverse biological processes through five receptor types, S1P(1-5). Two or more S1P receptors are usually co-expressed on target cells. We have previously shown that smooth muscle cells of the gut co-express S1P(1) and S1P(2) receptors that could mediate distinct functions. In the absence of selective agonists and antagonists, we developed siRNA constructs to silence each receptor separately. The constructs were based on identical sequences in several mammalian species. A lentiviral vector-based system was used to deliver siRNA into HEK293T cells and smooth muscle cells. One S1P(1) and two S1P(2) siRNA constructs specifically inhibited ectopic expression of S1P(1) and S1P(2) receptors, respectively, as determined by immunocytochemistry and Western blot, and endogenous expression of S1P(1) and S1P(2) receptors in smooth muscle cells, as determined by RT-PCR. Measurement of PLC-beta and Rho kinase activities, which mediate initial and sustained muscle contraction, confirmed receptor silencing and showed that contraction is mediated exclusively by S1P(2) receptors.     

Akt1K64/276与SUMO1结合激活ERK1/2-Elk1-BDNF信号通路

本研究主要从蛋白质结构分析Akt1 SUMO化的位点及位点的突变对其结构与功能的影响。采用多种软件分析Akt1 SUMO化位点和Akt1野生型(Akt1wt)及Akt1K64/276R的理化性质、亲/疏水性及二/三级结构;分析结果显示,Akt1K64/276R较Akt1wt,亲/疏水性未改变,α-螺旋和β-折叠都有少量的不同。三级结构分析显示,与野生型组相比,Akt1K64R氢键增多。以Myc-Akt1wt-pcDNA3.1为模板,采用PCR定点突变技术扩增出Myc-Akt1K64/276R。DNA序列分析结果显示,Myc-Akt1K64/276R基因序列编码赖氨酸(K)的密码子AAG被成功突变为精氨酸(R)密码子AGG。免疫沉淀和免疫印迹结果显示,不共转PIAS3,Akt1也能与SUMO1结合;Myc-Akt1wt和Myc-Akt1K64/276R均可在HEK293细胞中高效表达;转染Myc-Akt1K64/276R组SUMO化水平降低了70%左右(P<0.05)。免疫印迹结果显示,在小鼠海马神经细胞HT22中,Myc-Akt1wt组ERK1/2磷酸化水平及BDNF蛋白水平是突...     

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.     

Total synthesis of the mollu-series glycosyl ceramides alpha-D-Manp-(1----3)-beta-D-Manp-(1----4)-beta-D-Glcp-(1----1)-Cer and alpha-D-Manp-(1----3)-[beta-D-Xylp-(1----2)]-beta-D-Manp-(1----4)-beta- D-Glcp-(1----1)-Cer

The mollu-series glycosphingolipids, O-alpha-D-mannopyranosyl-(1----3)-O-beta-D-mannopyranosyl-(1----4)-O-bet a-D-glucopyranosyl-(1----1)-2-N-tetracosanoyl-(4E)-sphingeni ne and O-alpha-D-mannopyranosyl-(1----3)-O-[beta-D-xylopyranosyl-(1----2])-O- beta-D-mannopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-2-N- tetracosanoyl-(4E)-sphingenine, were synthesized for the first time by using 2,3,4-tri-O-acetyl-D-xylopyranosyl trichloroacetimidate, methyl 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranoside, benzyl O-(4,6-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside 9, and (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol 6 as the key intermediates. The hexa-O-benzyl disaccharide 9 was prepared by coupling two monosaccharide synthons, namely, 2,3-di-O-allyl-4,6-di-O-benzyl-alpha-D-mannopyranosyl bromide and benzyl 2,3,6-tri-O-benzyl-beta-D-glucopyranoside. It was demonstrated that azide 6 was highly efficient as a synthon for the ceramide part in the coupling with both glycotriaosyl and glycotetraosyl donors, particularly in the presence of trimethylsilyl triflate.     

Anti N1 Cross-Protecting Antibodies Against H5N1 Detected in H1N1 Infected People

The A(H5N1) influenza virus pandemic may be the result of avian H5N1 adapting to humans, leading to massive human to human transmission in a context of a lack of pre-existing immunity. As A(H1N1) and A(H5N1) share the same neuraminidase subtype, anti-N1 antibodies subsequent to H1N1 infections or vaccinations may confer some protection against A(H5N1). We analysed, by microneutralization assay, the A/Vietnam/1194/04 (H5N1) anti-N1 cross-protection acquired either during A/NewCaledonia/20/99 (H1N1) infection or vaccination. In cases with documented H1N1 infection, H5N1 cross-protection could be observed only in patients born between 1930 and 1950. No such protection was detected in the sera of vaccinated individuals.     

Slo1 caveolin-binding motif, a mechanism of caveolin-1-Slo1 interaction regulating Slo1 surface expression

The large conductance, voltage- and Ca2+-activated potassium (MaxiK, BK) channel and caveolin-1 play important roles in regulating vascular contractility. Here, we hypothesized that the MaxiK alpha-subunit (Slo1) and caveolin-1 may interact with each other. Slo1 and caveolin-1 physiological association in native vascular tissue is strongly supported by (i) detergent-free purification of caveolin-1-rich domains demonstrating a pool of aortic Slo1 co-migrating with caveolin-1 to light density sucrose fractions, (ii) reverse co-immunoprecipitation, and (iii) double immunolabeling of freshly isolated myocytes revealing caveolin-1 and Slo1 proximity at the plasmalemma. In HEK293T cells, Slo1-caveolin-1 association was unaffected by the smooth muscle MaxiK beta1-subunit. Sequence analysis revealed two potential caveolin-binding motifs along the Slo1 C terminus, one equivalent, 1007YNMLCFGIY1015, and another mirror image, 537YTEYLSSAF545, to the consensus sequence, varphiXXXXvarphiXXvarphi. Deletion of 1007YNMLCFGIY1015 caused approximately 80% loss of Slo1-caveolin-1 association while preserving channel normal folding and overall Slo1 and caveolin-1 intracellular distribution patterns. 537YTEYLSSAF545 deletion had an insignificant dissociative effect. Interestingly, caveolin-1 coexpression reduced Slo1 surface and functional expression near 70% without affecting channel voltage sensitivity, and deletion of 1007YNMLCFGIY1015 motif obliterated channel surface expression. The results suggest 1007YNMLCFGIY1015 possible participation in Slo1 plasmalemmal targeting and demonstrate its role as a main mechanism for caveolin-1 association with Slo1 potentially serving a dual role: (i) maintaining channels in intracellular compartments downsizing their surface expression and/or (ii) serving as anchor of plasma membrane resident channels to caveolin-1-rich membranes. Because the caveolin-1 scaffolding domain is juxtamembrane, it is tempting to suggest that Slo1-caveolin-1 interaction facilitates the tethering of the Slo1 C-terminal end to the membrane.     

Regulation of limb development by the sphingosine 1-phosphate receptor S1p1/EDG-1 occurs via the hypoxia/VEGF axis

Angiogenesis, also known as new blood vessel formation, is regulated coordinately with other tissue differentiation events during limb development. Although vascular endothelial cell growth factor (VEGF) is important in the regulation of angiogenesis, chondrogenesis and osteogenesis during limb development, the role of other angiogenic factors is not well understood. Sphingosine 1-phosphate, a platelet-derived lipid mediator, regulates angiogenesis and vascular maturation via its action on the G-protein-coupled receptor S1P(1) (also known as EDG-1). In addition to vascular defects, abnormal limb development was also observed in S1p(1)(-/-) mice. Here we show that strong induction of S1P(1) expression is observed in the blood vessels and the interdigital mesenchymal cells during limb development. Deletion of S1P(1) results in aberrant chondrocyte condensation and defective digit morphogenesis. Interestingly, the vasculature in the S1p(1)(-/-) limbs was hyperplastic and morphologically altered. In addition, the hypoxia inducible factor (HIF)-1 alpha and its response gene VEGF were induced in S1p(1)(-/-) limbs. However, aberrant regulation of HIF-1 alpha and VEGF were not observed in embryonic fibroblasts derived from S1p(1)(-/-) mice, suggesting a non-cell autonomous effect of S1P(1) on VEGF expression. Indeed, similar limb defects were observed in endothelium-specific S1P(1) null mice in vivo. These data suggest that the function of S1P(1) in the developing vasculature is essential for proper limb development.     

Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca(2+) sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca(2+) entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca(2+) entry and activated typical I(CRAC) current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I(SOC), that was blocked by 1 microm Gd(3+) and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I(SOC) in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I(SOC), whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.     

AXR1-ECR1 and AXL1-ECR1 heterodimeric RUB-activating enzymes diverge in function in Arabidopsis thaliana

RELATED TO UBIQUITIN (RUB) modification of CULLIN (CUL) subunits of the CUL-RING ubiquitin E3 ligase (CRL) superfamily regulates CRL ubiquitylation activity. RUB modification requires E1 and E2 enzymes that are analogous to, but distinct from, those activities required for UBIQUITIN (UBQ) attachment. Gene duplications are widespread in angiosperms, and in line with this observation, components of the RUB conjugation pathway are found in multiples in Arabidopsis. To further examine the extent of redundancy within the RUB pathway, we undertook biochemical and genetic characterizations of one such duplication event- the duplication of the genes encoding a subunit of the RUB E1 into AUXIN RESISTANT1 (AXR1) and AXR1-LIKE1 (AXL1). In vitro, the two proteins have similar abilities to function with E1 C-TERMINAL-RELATED1 (ECR1) in catalyzing RUB1 activation and RUB1-ECR1 thioester formation. Using mass spectrometry, endogenous AXR1 and AXL1 proteins were found in complex with 3HA-RUB1, suggesting that AXR1 and AXL1 exist in parallel RUB E1 complexes in Arabidopsis. In contrast, AXR1 and AXL1 differ in ability to correct phenotypic defects in axr1-30, a severe loss-of-function AXR1 mutant, when the respective coding sequences are expressed from the same promoter, suggesting differential in vivo functions. These results suggest that while both proteins function in the RUB pathway and are biochemically similar in RUB-ECR1 thioester formation, they are not functionally equivalent.     

Nucleic acid binding and unfolding properties of ribosomal protein S1 and the derivatives S1-F1 and m1-S1.

The nucleic acid binding and unwinding properties of wild-type Escherichia coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol. 127, 41-45 (1979) and J. Biol. Chem. 254, 4309-4312 (1979). The mutant (m1-S1) contains 77% and the fragment (S1-F1) 66% of the polypeptide chain length (approximately 600 amino acid residues) of protein S1. The mutant is active in protein synthesis in vitro; the fragment, although retaining one or more of the functional domains of S1, is inactive in protein synthesis. We find that m1-S1 is is almost as effective as S1 in binding to poly(rU), phage MS2 RNA and simian virus 40 (SV40) DNA, and in unfolding poly(rU) and the helical structures present in MS2 RNA and phi X174 viral DNA. S1-F1, however, binds to poly(rU) and denatured SV40 DNA, but not to MS2 RNA. It unfolds neither poly(rU), nor the residual secondary structure of MS2 RNA or phi X174 viral DNA. Thus, there appears to be a correlation between the loss in ability of S1 to unwind RNA and the loss in its ability to function in protein synthesis.     

Aldehyde dehydrogenase 1B1 (ALDH1B1) is a potential biomarker for human colon cancer

Aldehyde dehydrogenases (ALDHs) belong to a superfamily of NAD(P)⁺-dependent enzymes, which catalyze the oxidation of endogenous and exogenous aldehydes to their corresponding acids. Increased expression and/or activity of ALDHs, particularly ALDH1A1, have been reported to occur in human cancers. It is proposed that the metabolic function of ALDH1A1 confers the “stemness” properties to normal and cancer stem cells. Nevertheless, the identity of ALDH isozymes that contribute to the enhanced ALDH activity in specific types of human cancers remains to be elucidated. ALDH1B1 is a mitochondrial ALDH that metabolizes a wide range of aldehyde substrates including acetaldehyde and products of lipid peroxidation (LPO). In this study, we immunohistochemically examined the expression profile of ALDH1A1 and ALDH1B1 in human adenocarcinomas of colon (N = 40), lung (N = 30), breast (N = 33) and ovary (N = 33) using an NIH tissue array. The immunohistochemical expression of ALDH1A1 or ALDH1B1 in tumor tissues was scored by their intensity (scale = 1–3) and extensiveness (% of total cancer cells). Herein we report a 5.6-fold higher expression score for ALDH1B1 in cancerous tissues than that for ALDH1A1. Remarkably, 39 out of 40 colonic cancer specimens were positive for ALDH1B1 with a staining intensity of 2.8 ± 0.5. Our study demonstrates that ALDH1B1 is more profoundly expressed in the adenocarcinomas examined in this study relative to ALDH1A1 and that ALDH1B1 is dramatically upregulated in human colonic adenocarcinoma, making it a potential biomarker for human colon cancer.