Properties of N-Acetylhistamine Deacetylase in Mammalian Brain

Properties of N-acetylhistamine deacetylase in rat brain were studied, utilizing a sensitive coupled radioenzymatic assay. The Km for N-acetylhistamine for this deacetylase was 660 microM and its Vmax was 330 pmol/h/mg protein. N-Acetylhistamine deacetylase activity increased 80% in the presence of 1 mM Mn2+. The Km of Mn2+ was 40 microM. The enzyme is primarily a soluble enzyme with a relatively uniform regional distribution, unlike the distribution for histamine and histidine decarboxylase. Neonatal activity of this enzyme in rat brain is higher than in adult brain. alpha-Fluoromethylhistidine does not affect the activity of N-acetylhistamine, indicating that deacetylation probably does not play a regulatory role in the synthesis of brain histamine.     

Altered modulation of lamin A/C‐HDAC2 interaction and p21 expression during oxidative stress response in HGPS

Defects in stress response are main determinants of cellular senescence and organism aging. In fibroblasts from patients affected by Hutchinson–Gilford progeria, a severe LMNA‐linked syndrome associated with bone resorption, cardiovascular disorders, and premature aging, we found altered modulation of CDKN1A, encoding p21, upon oxidative stress induction, and accumulation of senescence markers during stress recovery. In this context, we unraveled a dynamic interaction of lamin A/C with HDAC2, an histone deacetylase that regulates CDKN1A expression. In control skin fibroblasts, lamin A/C is part of a protein complex including HDAC2 and its histone substrates; protein interaction is reduced at the onset of DNA damage response and recovered after completion of DNA repair. This interplay parallels modulation of p21 expression and global histone acetylation, and it is disrupted by LMNAmutations leading to progeroid phenotypes. In fact, HGPS cells show impaired lamin A/C‐HDAC2 interplay and accumulation of p21 upon stress recovery. Collectively, these results link altered physical interaction between lamin A/C and HDAC2 to cellular and organism aging. The lamin A/C‐HDAC2 complex may be a novel therapeutic target to slow down progression of progeria symptoms.     

Activation of Class I histone deacetylases contributes to mitochondrial dysfunction in cardiomyocytes with altered complex activities

Histone deacetylases (HDACs) play vital roles in the pathophysiology of heart failure, which is associated with mitochondrial dysfunction. Tumor necrosis factor-α (TNF-α) contributes to the genesis of heart failure and impairs mitochondria. This study evaluated the role of HDACs in TNF-α-induced mitochondrial dysfunction and investigated their therapeutic potential and underlying mechanisms. We measured mitochondrial oxygen consumption rate (OCR) and ATP production using Seahorse XF24 extracellular flux analyzer and bioluminescent assay in control and TNF-α (10 ng/ml, 24 h)-treated HL-1 cells with or without HDAC inhibition. TNF-α increased Class I and II (but not Class IIa) HDAC activities (assessed by Luminescent) with enhanced expressions of Class I (HDAC1, HDAC2, HDAC3, and HDAC8) but not Class IIb HDAC (HDAC6 and HDAC10) proteins in HL-1 cells. TNF-α induced mitochondrial dysfunction with impaired basal, ATP-linked, and maximal respiration, decreased cellular ATP synthesis, and increased mitochondrial superoxide production (measured by MitoSOX red fluorescence), which were rescued by inhibiting HDACs with MPT0E014 (1 μM, a Class I and IIb inhibitor), or MS-275 (1 μM, a Class I inhibitor). MPT0E014 reduced TNF-α-decreased complex I and II enzyme (but not III or IV) activities (by enzyme activity microplate assays). Our results suggest that Class I HDAC actions contribute to TNF-α-induced mitochondrial dysfunction in cardiomyocytes with altered complex I and II enzyme regulation. HDAC inhibition improves dysfunctional mitochondrial bioenergetics with attenuation of TNF-α-induced oxidative stress, suggesting the therapeutic potential of HDAC inhibition in cardiac dysfunction.     

Loss of α-Tubulin Acetylation Is Associated with TGF-β-induced Epithelial-Mesenchymal Transition

The epithelial-to-mesenchymal transition (EMT) is a process by which differentiated epithelial cells reprogram gene expression, lose their junctions and polarity, reorganize their cytoskeleton, increase cell motility and assume a mesenchymal morphology. Despite the critical functions of the microtubule (MT) in cytoskeletal organization, how it participates in EMT induction and maintenance remains poorly understood. Here we report that acetylated α-tubulin, which plays an important role in microtubule (MT) stabilization and cell morphology, can serve as a novel regulator and marker of EMT. A high level of acetylated α-tubulin was correlated with epithelial morphology and it profoundly decreased during TGF-β-induced EMT. We found that TGF-β increased the activity of HDAC6, a major deacetylase of α-tubulin, without affecting its expression levels. Treatment with HDAC6 inhibitor tubacin or TGF-β type I receptor inhibitor SB431542 restored the level of acetylated α-tubulin and consequently blocked EMT. Our results demonstrate that acetylated α-tubulin can serve as a marker of EMT and that HDAC6 represents an important regulator during EMT process.     

Homodimeric PHD Domain-containing Rco1 Subunit Constitutes a Critical Interaction Hub within the Rpd3S Histone Deacetylase Complex

Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way.