第二位抑制器突变显示了人类P-糖蛋白 (ABCB1) 的药物结合口袋和核酸结合域1之间的联系
Megumi Murakami1, Andaleeb Sajid1, Sabrina Lusvarghi1
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
概括
研究人类P-糖蛋白 (P-gp) 突变揭示了特定残留物变化如何影响药物运输. 第二位点突变可以恢复P-gp的功能,突出了对理解化学抵抗至关重要的通信通路.
科学领域:
- 生物化学和分子生物学
- 癌症研究 癌症研究
- 药理学 药理学 是一个学科.
背景情况:
- 人类P-糖蛋白 (P-gp/ABCB1) 在癌症中过度表达,通过排放化疗药物,导致多药性耐药性.
- 了解P-gp的结构-功能关系对于开发克服化学抵抗的策略至关重要.
- 之前的研究发现,在基质运输所必需的P-gp药物结合口袋中,有15种保存残留物.
研究的目的:
- 为了研究在基质运输中人类P-gp的跨膜域 (TMDs) 1和2中的特定残留物的作用.
- 为了识别能够恢复受损药物流动的P-gp突变体的运输功能的第二位移位突变.
- 阐明 P-gp 中基质结合口袋和核酸结合域 (NBD) 之间的通信通路.
主要方法:
- 产生和表达两种新的P-gp突变:6Y (在TMD1中有6个替代物) 和9Y (在TMD2中有9个替代物).
- 用各种基质对野生型和突变的P-gp蛋白进行基质运输的功能性评估.
- 识别了恢复6Y突变功能的第二位抑制突变 (细胞内螺旋4中的F916Y和NBD1 Q循环中的F480Y).
- 生物化学测试和分子动力学模拟来分析NBD1与基质结合部位之间的通信.
主要成果:
- 6Y突变显示出显著受损的基质运输,而9Y突变表现出野生类型的流体活动.
- 第二位点突变F916Y和F480Y成功地恢复了非运输6Y突变的运输功能.
- 分子动力学模拟表明NBD1 Q循环与基质结合部位之间通过细胞内螺旋4进行通信.
结论:
- 在TMD1中的特定残留物对P-gp的广泛基板运输能力至关重要.
- 这项研究提供了人类P-gp中第二位抑制剂的第一个证据,可以在初级突变后挽救运输功能.
- 这些发现揭示了一条涉及NBD1和细胞内螺旋4的通信通路,这对P-gp功能至关重要,并可能涉及其他ABC载体.
相关概念视频
01:49Conserved Binding Sites
4.2K
Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
4.2K
01:27ABC Transporters: Importer
2.7K
ATP-binding cassette or ABC transporters are a class of ATP-driven pumps that hydrolyze ATP to move solutes across the membrane. They can be grouped into importers and exporters. While exporters are present in all domains of life, importers exist only in bacteria and some plants.
In bacteria, based on the number of transmembrane helices and the chemical nature of their substrates, the ABC importers can be divided into three types:
In bacteria, based on the number of transmembrane helices and the chemical nature of their substrates, the ABC importers can be divided into three types:
2.7K
01:31ABC Transporters: Exporter
4.4K
ATP-binding cassette or ABC transporter is the largest superfamily of integral membrane proteins. The transporters have transmembrane-binding domains (TMDs) and nucleotide-binding domains (NBDs). The TMDs are specific to their substrates, whereas the NBDs are similar to engines that complete ATP hydrolysis to complete the substrate transport. They can be full transporters consisting of two TMDs and NBDs, half transporters with one TMD and NBD, while some encoded with a single TMD or NBD are...
4.4K
01:19Allosteric Proteins-ATCase
5.8K
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
5.8K
00:49Ligand Binding and Linkage
4.8K
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
4.8K
02:27Nonsense-mediated mRNA Decay
10.7K
The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
10.7K