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The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity
Biochemistry ( IF 2.9 ) Pub Date : 2017-12-14 00:00:00 , DOI: 10.1021/acs.biochem.7b00936 Matthew J. Ranaghan 1 , Michael A. Durney 1 , Michael F. Mesleh 1 , Patrick R. McCarren 1 , Colin W. Garvie 1 , Douglas S. Daniels 2 , Kimberly L. Carey 1 , Adam P. Skepner 1 , Beth Levine 3 , Jose R. Perez 1
Biochemistry ( IF 2.9 ) Pub Date : 2017-12-14 00:00:00 , DOI: 10.1021/acs.biochem.7b00936 Matthew J. Ranaghan 1 , Michael A. Durney 1 , Michael F. Mesleh 1 , Patrick R. McCarren 1 , Colin W. Garvie 1 , Douglas S. Daniels 2 , Kimberly L. Carey 1 , Adam P. Skepner 1 , Beth Levine 3 , Jose R. Perez 1
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Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3–5 mg/L in an Escherichia coli culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (KD ∼ 0.45 μM) that interacts with Bcl-2 (KD = 4.3 ± 1.2 μM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer KD 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold.
中文翻译：
自噬相关的Beclin-1蛋白需要螺旋线圈和BARA结构域才能形成具有亚微摩尔亲和力的均聚物。
Beclin-1（BECN1）是巨噬细胞自噬的重要组成部分。此过程是高度保守的生存机制，该机制通过将受损的细胞成分或病原体封装在与溶酶体融合的双层囊泡中来回收受损的细胞成分或病原体，从而使囊泡内容物降解。BECN1的突变或表达谱改变与多种癌症和神经退行性疾病有关。还已知包括HIV和单纯疱疹病毒1（HSV-1）在内的病毒会专门针对BECN1作为逃避宿主防御机制的手段。自噬受BECN1和Bcl-2之间的相互作用调节，Bcl-2是凋亡通路中的一种前生存蛋白，可稳定BECN1同型二聚体。通过磷酸化或竞争性结合破坏同型二聚体通过未知机制促进自噬。大肠杆菌培养）和全长人BECN1的表征。我们的分析表明，全长BECN1存在作为可溶性同二聚体（ķ d〜0.45μM），与Bcl-2的（相互作用ķ d = 4.3±1.2μM），并结合于脂质膜。提出二聚化是由BECN1的卷曲螺旋区域介导的。缺少C末端BARA结构域但包括卷曲螺旋区的构建体表现出同源二聚体K D。比全长BECN1弱3.5倍，表明BEA1的BARA结构域和卷曲螺旋区域均有助于形成二聚体。使用定点诱变，我们显示先前显示与BARA结构域相互作用的卷曲螺旋区域C端残基在二聚化中起关键作用，并且突变使界面减弱约5倍。
更新日期：2017-12-14
中文翻译：
自噬相关的Beclin-1蛋白需要螺旋线圈和BARA结构域才能形成具有亚微摩尔亲和力的均聚物。
Beclin-1（BECN1）是巨噬细胞自噬的重要组成部分。此过程是高度保守的生存机制，该机制通过将受损的细胞成分或病原体封装在与溶酶体融合的双层囊泡中来回收受损的细胞成分或病原体，从而使囊泡内容物降解。BECN1的突变或表达谱改变与多种癌症和神经退行性疾病有关。还已知包括HIV和单纯疱疹病毒1（HSV-1）在内的病毒会专门针对BECN1作为逃避宿主防御机制的手段。自噬受BECN1和Bcl-2之间的相互作用调节，Bcl-2是凋亡通路中的一种前生存蛋白，可稳定BECN1同型二聚体。通过磷酸化或竞争性结合破坏同型二聚体通过未知机制促进自噬。大肠杆菌培养）和全长人BECN1的表征。我们的分析表明，全长BECN1存在作为可溶性同二聚体（ķ d〜0.45μM），与Bcl-2的（相互作用ķ d = 4.3±1.2μM），并结合于脂质膜。提出二聚化是由BECN1的卷曲螺旋区域介导的。缺少C末端BARA结构域但包括卷曲螺旋区的构建体表现出同源二聚体K D。比全长BECN1弱3.5倍，表明BEA1的BARA结构域和卷曲螺旋区域均有助于形成二聚体。使用定点诱变，我们显示先前显示与BARA结构域相互作用的卷曲螺旋区域C端残基在二聚化中起关键作用，并且突变使界面减弱约5倍。
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