LAS17
目录号 : GC63518
LAS17 是一种强效的，不可逆的，选择性谷胱甘肽 S-转移酶 Pi (GSTP1) 抑制剂。LAS17 抑制 GSTP1 活性，IC50 为 0.5 µM.
Cas No.:2362527-67-9
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
LAS17 is a potent and selective tyrosine-directed irreversible inhibitor for glutathione S-Transferase Pi (GSTP1) [1]. LAS17 inhibits GSTP1 activity with an IC50 of 0.5 µM[2].
Glutathione S-Transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. LAS17 inhibits GSTP1 activity in vitro in a concentration-dependent manner[1]. LAS17 (10 µ Serum-free survival 48 h) treatment in 231MFP breast cancer cells recapitulates the serum-free cell survival impairments observed with genetic inactivation of GSTP1[2]. GSTP1 knockdown in LAS17 (10 µM) treatment in 231MFP cells results in increased levels of phosphorylated AMPK and acetyl CoA carboxylase (ACC)[2].LAS17 treatment in 231MFP cells also shows reduced levels of ATP, lactic acid, purine nucleotides, and diacylated phospholipids and alkylacyl ether lipids and increased levels of acyl carnitines (ACs), ceramides, lysophospholipids[2].
Daily administration of LAS17 (20 mg/kg ip, once per day) significantly impairs 231MFP breast tumor xenograft growth in immune-deficient mice when treatment is initiated 2 days after subcutaneous injection of cells, and LAS17 even slows tumor growth when initiated 16 days after tumor implantation, with no observable toxicity and no weight-change[2].
[1]. L A Crawford, et al. A tyrosine-reactive irreversible inhibitor for glutathione S-transferase Pi (GSTP1). Mol Biosyst. 2016 May 24;12(6):1768-71.
[2]. Sharon M Louie, et al. GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity. Cell Chem Biol. 2016 May 19;23(5):567-578.
| Cas No. | 2362527-67-9 | SDF | |
| 分子式 | C15H20Cl2N4O2 | 分子量 | 359.25 |
| 溶解度 | 储存条件 | Store at -20°C | |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.7836 mL | 13.9179 mL | 27.8358 mL |
| 5 mM | 0.5567 mL | 2.7836 mL | 5.5672 mL |
| 10 mM | 0.2784 mL | 1.3918 mL | 2.7836 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
A second LAS17 monomeric actin-binding motif functions in Arp2/3-dependent actin polymerization during endocytosis
Traffic 2015 Apr;16(4):379-97.PMID:25615019DOI:10.1111/tra.12259.
During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (LAS17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in LAS17. Here, we describe the in vitro and in vivo characterization of such LAS17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known LAS17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in LAS17 and advance our knowledge on the mechanism of actin assembly during CME.
Phosphorylation of the WH2 domain in yeast LAS17/WASP regulates G-actin binding and protein function during endocytosis
Sci Rep 2021 May 6;11(1):9718.PMID:33958621DOI:10.1038/s41598-021-88826-z.
Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. WH2 domains are short sequence motifs found in many different actin binding proteins including WASP family proteins which regulate the actin nucleating complex Arp2/3. In this study we reveal a phosphorylation site, Serine 554, within the WH2 domain of the yeast WASP homologue LAS17. Both phosphorylation and a phospho-mimetic mutation reduce actin monomer binding affinity while an alanine mutation, generated to mimic the non-phosphorylated state, increases actin binding affinity. The effect of these mutations on the Las17-dependent process of endocytosis in vivo was analysed and leads us to propose that switching of LAS17 phosphorylation states may allow progression through distinct phases of endocytosis from site assembly through to the final scission stage. While the study is focused on LAS17, the sole WASP family protein in yeast, our results have broad implications for our understanding of how a key residue in this conserved motif can underpin the many different actin regulatory roles with which WH2 domains have been associated.
SLAC, a complex between Sla1 and LAS17, regulates actin polymerization during clathrin-mediated endocytosis
Mol Biol Cell 2012 Nov;23(21):4256-72.PMID:22973053DOI:10.1091/mbc.E11-12-1022.
During clathrin-mediated endocytosis, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. LAS17 (yeast WASp) is the strongest activator of the Arp2/3 complex in yeast cells; it is not autoinhibited and arrives to endocytic sites 20 s before actin polymerization begins. It is unclear how LAS17 is kept inactive for 20 s at endocytic sites, thus restricting actin polymerization to late stages of endocytosis. In this paper, we demonstrate that LAS17 is part of a large and biochemically stable complex with Sla1, a clathrin adaptor that inhibits LAS17 activity. The interaction is direct, multivalent, and strong, and was mapped to novel LAS17 polyproline motifs that are simultaneously class I and class II. In vitro pyrene-actin polymerization assays established that Sla1 inhibition of LAS17 activity depends on the class I/II LAS17 polyproline motifs and is based on competition between Sla1 and monomeric actin for binding to LAS17. Furthermore, live-cell imaging showed the interaction with Sla1 is important for normal LAS17 recruitment to endocytic sites, inhibition during the initial 20 s, and efficient endocytosis. These results advance our understanding of the regulation of actin polymerization in endocytosis.
A novel actin-binding motif in LAS17/WASP nucleates actin filaments independently of Arp2/3
Curr Biol 2013 Feb 4;23(3):196-203.PMID:23290554DOI:10.1016/j.cub.2012.12.024.
Background: Actin nucleation is the key rate-limiting step in actin polymerization, and tight regulation of this process is critical to ensure that actin filaments form only at specific regions of the cell. LAS17 is the primary activator of Arp2/3-driven actin nucleation in yeast and is required for membrane invagination during endocytosis. Its mammalian homolog, WASP, has also been studied extensively as an activator of Arp2/3-driven actin polymerization. In both LAS17 and WASP, actin nucleation activity is attributed to an ability to bind actin through a WH2 domain and to bind Arp2/3 through an acidic region. The central region of both LAS17 and WASP is rich in proline residues and is generally considered to bind to SH3-domain-containing proteins. Results: We have identified a novel actin-binding activity in the polyproline domain of both yeast LAS17 and mammalian WASP. The polyproline domain of LAS17 is also able to nucleate actin filaments independently of Arp2/3. Mutational analysis reveals that proline residues are required for this nucleation activity and that the binding site on actin maps to a region distinct from those used by other nucleation activities. In vivo analysis of yeast strains expressing LAS17 mutated in the WH2 domain, one of its proline motifs, or both shows additive defects in actin organization and endocytosis, with the proline mutant conferring more severe phenotypes than the WH2 mutant. Conclusions: Our data demonstrate a new actin-binding and nucleation mechanism in LAS17/WASP that is required for its function in actin regulation during endocytosis.
Lsb1 is a negative regulator of LAS17 dependent actin polymerization involved in endocytosis
PLoS One 2013;8(4):e61147.PMID:23577202DOI:10.1371/journal.pone.0061147.
The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott-Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, LAS17, that requires additional factors for its regulation. Lsb1 and Lsb2/Pin3 are two yeast homologous proteins bearing an SH3 domain that were identified as Las17-binding proteins. Lsb2/Pin3 that promotes prion induction was suggested to link this prion formation to the actin cytoskeleton. However, the cellular role of Lsb1 and the molecular function of both Lsb1 and Lsb2 remain unknown. In this study, we show that Lsb1 and/or Lsb2 full-length proteins inhibit Las17-mediated actin polymerization in vitro, Lsb2 being a less potent inhibitor of LAS17 activity compared to Lsb1. Addition of Lsb1 or Lsb2 to the corresponding full-length Lsb1/2 further inhibits LAS17 activity. Lsb1 and Lsb2 form homo- and hetero-oligomeric complexes suggesting that these two proteins could regulate LAS17 activity via dimerization or cooperative binding. In vivo, overexpressed Lsb1 and Lsb2 proteins cluster Las17-CFP in few cytoplasmic punctate structures that are also positive for other Arp2/3-dependent actin polymerization effectors like Sla1 or Abp1. But, only Lsb1 overexpression blocks the internalization step of receptor-mediated endocytosis. This shows a specific function of Lsb1 in endocytosis.