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201.
202.
203.
Homing endonucleases typically contain one of four conserved catalytic motifs, and other elements that confer tight DNA binding. I-CreII, which catalyzes homing of the Cr.psbA4 intron, is unusual in containing two potential catalytic motifs, H-N-H and GIY-YIG. Previously, we showed that cleavage by I-CreII leaves ends (2-nt 3′ overhangs) that are characteristic of GIY-YIG endonucleases, yet it has a relaxed metal requirement like H-N-H enzymes. Here we show that I-CreII can bind DNA without an added metal ion, and that it binds as a monomer, akin to GIY-YIG enzymes. Moreover, cleavage of supercoiled DNA, and estimates of strand-specific cleavage rates, suggest that I-CreII uses a sequential cleavage mechanism. Alanine substitution of a number of residues in the GIY-YIG motif, however, did not block cleavage activity, although DNA binding was substantially reduced in several variants. Substitution of conserved histidines in the H-N-H motif resulted in variants that did not promote DNA cleavage, but retained high-affinity DNA binding—thus identifying it as the catalytic motif. Unlike the non-specific H-N-H colicins, however; substitution of the conserved asparagine substantially reduced DNA binding (though not the ability to promote cleavage). These results indicate that, in I-CreII, two catalytic motifs have evolved to play important roles in specific DNA binding. The data also indicate that only the H-N-H motif has retained catalytic ability. 相似文献
204.
Constantine G. Boojamra Jay P. Parrish David Sperandio Ying Gao Oleg V. Petrakovsky Sharon K. Lee David Y. Markevitch Jennifer E. Vela Genevieve Laflamme James M. Chen Adrian S. Ray Abraham C. Barron Mark L. Sparacino Manoj C. Desai Choung U. Kim Tomas Cihlar Richard L. Mackman 《Bioorganic & medicinal chemistry》2009,17(4):1739-1746
205.
Regulation of classic cadherins plays a critical role in tissue remodeling during development and cancer; however, less attention has been paid to the importance of desmosomal cadherins. We previously showed that EGFR inhibition results in accumulation of the desmosomal cadherin, desmoglein 2 (Dsg2), at cell-cell interfaces accompanied by inhibition of matrix metalloprotease (MMP)-dependent shedding of the Dsg2 ectodomain and tyrosine phosphorylation of its cytoplasmic domain. Here, we show that EGFR inhibition stabilizes Dsg2 at intercellular junctions by interfering with its accumulation in an internalized cytoplasmic pool. Furthermore, MMP inhibition and ADAM17 RNAi, blocked shedding and depleted internalized Dsg2, but less so E-cadherin, in highly invasive SCC68 cells. ADAM9 and 15 silencing also impaired Dsg2 processing, supporting the idea that this desmosomal cadherin can be regulated by multiple ADAM family members. In contrast, ADAM10 siRNA enhanced accumulation of a 100-kDa Dsg2 cleavage product and internalized pool of Dsg2. Although both MMP and EGFR inhibition increased intercellular adhesive strength in control cells, the response to MMP-inhibition was Dsg2-dependent. These data support a role for endocytic trafficking in regulating desmosomal cadherin turnover and function and raise the possibility that internalization and regulation of desmosomal and classic cadherin function can be uncoupled mechanistically. 相似文献
206.
E-Chiang Lee Urvi Desai Gennady Gololobov Seokjoo Hong Xiao Feng Xuan-Chuan Yu Jason Gay Nat Wilganowski Cuihua Gao Ling-Ling Du Joan Chen Yi Hu Sharon Zhao Laura Kirkpatrick Matthias Schneider Brian P. Zambrowicz Greg Landes David R. Powell William K. Sonnenburg 《The Journal of biological chemistry》2009,284(20):13735-13745
Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are
secreted proteins that regulate triglyceride (TG) metabolism in part by
inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of
wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4,
recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced
serum TG levels. In the present study, we mapped the region of mouse ANGPTL4
recognized by mAb 14D12 to amino acids
Gln29–His53, which we designate as specific
epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL,
consistent with its ability to neutralize the LPL-inhibitory activity of
ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence
similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino
acids Glu32–His55. We produced a mouse mAb against
this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the
binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL
activity in vitro. Treatment of wild-type as well as hyperlipidemic
mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the
lipid phenotype found in Angptl3-/- mice. These results
show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain
important for binding LPL and inhibiting its activity in vitro and
in vivo. Moreover, these results demonstrate that therapeutic
antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of
some forms of hyperlipidemia.Lipoprotein lipase
(LPL)5 plays a pivotal
role in lipid metabolism by catalyzing the hydrolysis of plasma triglycerides
(TGs). LPL is likely to be regulated by mechanisms that depend on nutritional
status and on the tissue in which it is expressed
(1–3).
Two secreted proteins, angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4
(ANGPTL4), play important roles in the regulation of LPL activity
(4,
5). ANGPTL3 and ANGPTL4 consist
of a signal peptide, an N-terminal segment containing coiled-coil domains, and
a C-terminal fibrinogen-like domain. The N-terminal segment as well as
full-length ANGPTL3 and ANGPTL4 have been shown to inhibit LPL activity, and
deletion of the N-terminal segment of ANGPTL3 and ANGPTL4 resulted in total
loss of LPL-inhibiting activity
(6,
7). These observations clearly
indicate that the N-terminal region of ANGPTL4 contains the functional domain
that inhibits LPL and affects plasma lipid levels. The coiled-coil domains
have been proposed to be responsible for oligomerization
(8); however, it is not known
whether the coiled-coil domains directly mediate the inhibition of LPL
activity.To define the physiological role of ANGPTL4 more clearly, we characterized
the pharmacological consequences of ANGPTL4 inhibition in mice treated with
the ANGPTL4-neutralizing monoclonal antibody (mAb) 14D12
(9). Injection of mAb 14D12
significantly lowered fasting TG levels in C57BL/6J mice relative to levels in
C57BL/6J mice treated with an isotype-matched anti-KLH control (KLH) mAb
(9). These reduced TG values
were similar to decreases in fasting plasma TG levels measured in
Angptl4 knock-out (-/-) mice. This study demonstrated that mAb 14D12
is a potent ANGPTL4-neutralizing antibody that is able to inhibit systemic
ANGPTL4 activity and thereby recapitulate the reduced lipid phenotype found in
Angptl4-/- mice. The readily apparent pharmacological
effect of mAb 14D12 prompted new questions about the epitope recognized by mAb
14D12 and how this antibody-antigen binding event affected ANGPTL4 function as
an LPL inhibitor.Although ANGPTL4 is able to interact directly with LPL
(10), it is not clear which
amino acids within ANGPTL4 mediate this interaction. Here we show that amino
acids Gln29–His53 of mANGPTL4 contain the epitope
for mAb 14D12. This region, hereby designated specific epitope 1 (SE1), also
defines a domain that mediates the interaction between ANGPTL4 and LPL and the
subsequent inactivation of LPL. With this information we present evidence that
ANGPTL3 also contains an SE1 region, and with antibodies specifically reactive
with ANGPTL3 SE1 we examine whether the ANGPTL3 SE1 region is involved in LPL
binding and inhibition. We also determined whether treatment of C57BL/6 mice
with an anti-ANGPTL3 SE1 mAb can recapitulate the phenotype of lower serum TG
and cholesterol levels found in Angptl3-/- mice. Finally
we tested the therapeutic potential of an anti-ANGPTL3 SE1 mAb for treatment
of hyperlipidemia in apolipoprotein E-/-
(ApoE-/-) or low density lipoprotein
receptor-/- (LDLr-/-) mice. 相似文献
207.
Liu J Desai KV Li Y Banu S Lee YK Qu D Heikkinen T Aaltonen K Muranen TA Kajiji TS Bonnard C Aittomäki K von Smitten K Blomqvist C Hopper JL Southey MC Brauch H;GENICA Consortium Chenevix-Trench G Beesley J Spurdle AB Chen X;Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer;Australian Ovarian Cancer Study Group Czene K Hall P Nevanlinna H Liu ET 《The HUGO journal》2009,3(1-4):31-40
208.
Cell-based three-dimensional systems are desirable in the field of high throughput screening assays due to their potential similarity to in vivo environment. We have used SH-SY5Y human neuroblastoma cells cultured in 3-D collagen hydrogel, confocal microscopy and immunofluorescence staining, to assess the merit of the system as a functional, cell-based biosensor. Our results show differences between 2-D and 3-D resting membrane potential development profile upon differentiation. There was no statistically significant difference in SH-SY5Y proliferation rate between 2-D monolayer and 3-D collagen culture formats. A large percentage of cells (2-D, 91.30% and 3-D, 84.93%) did not develop resting membrane potential value equal to or lower than -40 mV; instead cells exhibited a heterogeneous resting membrane potential distribution. In response to high K(+) (50 mM) depolarization, 3-D cells were less responsive in terms of increase in intracellular Ca(2+), in comparison to 2-D cells, supporting the hypothesis that 2-D cell calcium dynamics may be exaggerated. L-Type Ca(2+) expression levels based on staining results was inconsistent with Bay K 8644 channel activation results, strongly suggesting that either the majority of the channels were non-functional or could not be activated by Bay K 8644. In general, the results in this study confirm the depolarization-induced differences in intracellular calcium release when cultured using a 2-D versus a 3-D matrix. 相似文献
209.
Boys ML Schretzman LA Chandrakumar NS Tollefson MB Mohler SB Downs VL Penning TD Russell MA Wendt JA Chen BB Stenmark HG Wu H Spangler DP Clare M Desai BN Khanna IK Nguyen MN Duffin T Engleman VW Finn MB Freeman SK Hanneke ML Keene JL Klover JA Nickols GA Nickols MA Steininger CN Westlin M Westlin W Yu YX Wang Y Dalton CR Norring SA 《Bioorganic & medicinal chemistry letters》2006,16(4):839-844
We describe a series of 1,2,4-oxadiazoles, which are potent antagonists of the integrin alpha(v)beta3 and, in addition, show selectivity relative to the other beta3 integrin alpha(IIb)beta3. In whole cells, the majority of these analogs also demonstrated modest selectivity against other alpha(v) integrins such as alpha(v)beta1 and alpha(v)beta6. 相似文献
210.
Monien BH Henry BL Raghuraman A Hindle M Desai UR 《Bioorganic & medicinal chemistry》2006,14(23):7988-7998
Thrombin and factor Xa, two important procoagulant enzymes, have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. Our efforts on exploiting the indirect mechanism led us to study a carboxylic acid-based scaffold, which displayed major acceleration in the inhibition of these enzymes [J. Med. Chem.2005, 48, 1269, 5360]. This work advances the study to chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids, DHPs, which display interesting anticoagulant properties. Oligomers, ranging in size from tetramers to pentadecamers, were prepared through peroxidase-catalyzed oxidative coupling of caffeic, ferulic, and sinapic acids, and sulfated using triethylamine-sulfur trioxide complex. Chromatographic, spectroscopic, and elemental studies suggest that the DHPs are heterogeneous, polydisperse preparations composed of inter-monomer linkages similar to those found in natural lignins. Measurement of activated thromboplastin and prothrombin time indicates that both the sulfated and unsulfated derivatives of the DHPs display anticoagulant activity, which is dramatically higher than that of the reference polyacrylic acids. More interestingly, this activity approaches that of low-molecular-weight heparin with the sulfated derivative showing approximately 2- to 3-fold greater potency than the unsulfated parent. Studies on the inhibition of factor Xa and thrombin indicate that the oligomers exert their anticoagulant effect through both direct and indirect inhibition mechanisms. This dual inhibition property of 4-hydroxycinnamic acid-based DHP oligomers is the first example in inhibitors of coagulation. This work puts forward a novel, non-heparin structure, which may be exploited for the design of potent, dual action inhibitors of coagulation through combinatorial virtual screening on a library of DHP oligomers. 相似文献