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Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity.

Zhang L, Liu S, Liu N, Zhang Y, Liu M, Li D, Seto E, Yao TP, Shui W, Zhou J - Protein Cell (2014)

Bottom Line: However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates.Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1.These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, 300071, China.

ABSTRACT
Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.

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HDAC6 interacts with the new substrates. (A) 293T cell lysates were incubated with control IgG or anti-HDAC6 antibodies. The immunoprecipitates and cell lysates were then immunoblotted with the indicated antibodies. (B–D) 293T cells were transfected with GFP-HDAC6 or GFP alone. Anti-GFP immunoprecipitates and cell lysates were immunoblotted with the indicated antibodies. (E–G) Immunofluorescence confocal images of HeLa cells transfected with GFP-HDAC6 and stained with anti-MYH9 (E), anti-Hsc70 (F), or anti-DNAJA1 (G) antibodies and the DNA dye DAPI
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Fig3: HDAC6 interacts with the new substrates. (A) 293T cell lysates were incubated with control IgG or anti-HDAC6 antibodies. The immunoprecipitates and cell lysates were then immunoblotted with the indicated antibodies. (B–D) 293T cells were transfected with GFP-HDAC6 or GFP alone. Anti-GFP immunoprecipitates and cell lysates were immunoblotted with the indicated antibodies. (E–G) Immunofluorescence confocal images of HeLa cells transfected with GFP-HDAC6 and stained with anti-MYH9 (E), anti-Hsc70 (F), or anti-DNAJA1 (G) antibodies and the DNA dye DAPI

Mentions: Given that physical interactions are required for enzyme-substrate pairs as reported for HDAC6 and its known substrates (Hubbert et al., 2002; Zhang et al., 2003; Kovacs et al., 2005; Zhang et al., 2007), we examined by immunoprecipitation and immunofluorescence microscopy whether HDAC6 interacts with the new substrates. As shown in Fig. 3A, endogenous MYH9, Hsc70, and DNAJA1 were present in the immunoprecipitates of endogenous HDAC6 in 293T cells.Figure 3


Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity.

Zhang L, Liu S, Liu N, Zhang Y, Liu M, Li D, Seto E, Yao TP, Shui W, Zhou J - Protein Cell (2014)

HDAC6 interacts with the new substrates. (A) 293T cell lysates were incubated with control IgG or anti-HDAC6 antibodies. The immunoprecipitates and cell lysates were then immunoblotted with the indicated antibodies. (B–D) 293T cells were transfected with GFP-HDAC6 or GFP alone. Anti-GFP immunoprecipitates and cell lysates were immunoblotted with the indicated antibodies. (E–G) Immunofluorescence confocal images of HeLa cells transfected with GFP-HDAC6 and stained with anti-MYH9 (E), anti-Hsc70 (F), or anti-DNAJA1 (G) antibodies and the DNA dye DAPI
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4286133&req=5

Fig3: HDAC6 interacts with the new substrates. (A) 293T cell lysates were incubated with control IgG or anti-HDAC6 antibodies. The immunoprecipitates and cell lysates were then immunoblotted with the indicated antibodies. (B–D) 293T cells were transfected with GFP-HDAC6 or GFP alone. Anti-GFP immunoprecipitates and cell lysates were immunoblotted with the indicated antibodies. (E–G) Immunofluorescence confocal images of HeLa cells transfected with GFP-HDAC6 and stained with anti-MYH9 (E), anti-Hsc70 (F), or anti-DNAJA1 (G) antibodies and the DNA dye DAPI
Mentions: Given that physical interactions are required for enzyme-substrate pairs as reported for HDAC6 and its known substrates (Hubbert et al., 2002; Zhang et al., 2003; Kovacs et al., 2005; Zhang et al., 2007), we examined by immunoprecipitation and immunofluorescence microscopy whether HDAC6 interacts with the new substrates. As shown in Fig. 3A, endogenous MYH9, Hsc70, and DNAJA1 were present in the immunoprecipitates of endogenous HDAC6 in 293T cells.Figure 3

Bottom Line: However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates.Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1.These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, 300071, China.

ABSTRACT
Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.

Show MeSH
Related in: MedlinePlus