Histone deacetylases (HDACs) regulate myriad cellular processes by catalyzing the hydrolysis of acetyl–L-lysine residues in histone and nonhistone proteins. The Zn2+-dependent class IIb enzyme HDAC6 ...regulates microtubule function by deacetylating α-tubulin, which suppresses microtubule dynamics and leads to cell cycle arrest and apoptosis. Accordingly, HDAC6 is a target for the development of selective inhibitors that might be useful in new therapeutic approaches for the treatment of cancer, neurodegenerative diseases, and other disorders. Here, we present high-resolution structures of catalytic domain 2 from Danio rerio HDAC6 (henceforth simply “HDAC6”) complexed with compounds that selectively inhibit HDAC6 while maintaining nanomolar inhibitory potency: N-hydroxy-4-(N(2-hydroxyethyl)-2-phenylacetamido)methyl)-benzamide) (HPB), ACY-1215 (Ricolinostat), and ACY-1083. These structures reveal that an unusual monodentate Zn2+ coordination mode is exploited by sterically bulky HDAC6-selective phenylhydroxamate inhibitors. We additionally report the ultrahigh-resolution structure of the HDAC6–trichostatin A complex, which reveals two Zn2+-binding conformers for the inhibitor: a major conformer (70%) with canonical bidentate hydroxamate-Zn2+ coordination geometry and a minor conformer (30%) with monodentate hydroxamate-Zn2+ coordination geometry, reflecting a free energy difference of only 0.5 kcal/mol. The minor conformer is not visible in lower resolution structure determinations. Structural comparisons of HDAC6-inhibitor complexes with class I HDACs suggest active site features that contribute to the isozyme selectivity observed in biochemical assays.
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•Zn2+-histone deacetylases (HDACs) belong to the arginase-deacetylase superfamily.•HDACs hydrolyze acetyllysine, fatty acid acyllysine, and acetylpolyamine substrates.•HDACs are drug ...targets for the treatment of cancer and neurodegenerative disease.•Crystal structures of HDACs guide current drug design efforts.
Zinc-dependent histone deacetylases (HDACs) regulate the biological function of histone and non-histone proteins through the hydrolysis of acetyllysine side chains to yield free lysine and acetate. Certain HDAC isozymes exhibit alternative catalytic activities, such as polyamine deacetylase or lysine fatty acid deacylase activity. To date, crystal structures have been reported for class I HDACs (1–3, and 8), class IIa HDACs (4 and 7), and class IIb HDACs (6 and 10). Conserved active site residues mediate the chemistry of substrate activation and hydrolysis in these isozymes through a metal-activated water molecule assisted by general base–general acid catalysis. Upregulated HDAC activity is observed in cancer and neurodegenerative disease, and four HDAC inhibitors are currently approved for use in cancer chemotherapy. Crystal structures of HDAC-inhibitor complexes guide the design of new inhibitors with high affinity and selectivity for specific HDAC isozymes implicated in human disease.
Cationic polyamines such as spermidine and spermine are critical in all forms of life, as they regulate the function of biological macromolecules. Intracellular polyamine metabolism is regulated by ...reversible acetylation and dysregulated polyamine metabolism is associated with neoplastic diseases such as colon cancer, prostate cancer and neuroblastoma. Here we report that histone deacetylase 10 (HDAC10) is a robust polyamine deacetylase, using recombinant enzymes from Homo sapiens (human) and Danio rerio (zebrafish). The 2.85 Å-resolution crystal structure of zebrafish HDAC10 complexed with a transition-state analogue inhibitor reveals that a glutamate gatekeeper and a sterically constricted active site confer specificity for N
-acetylspermidine hydrolysis and disfavour acetyllysine hydrolysis. Both HDAC10 and spermidine are known to promote cellular survival through autophagy. Accordingly, this work sets a foundation for studying the chemical biology of autophagy through the structure-based design of inhibitors that may also serve as new leads for cancer chemotherapy.
Mineralization is a ubiquitous process in the animal kingdom and is fundamental to human development and health. Dysfunctional or aberrant mineralization leads to a variety of medical problems, and ...so an understanding of these processes is essential to their mitigation. Osteoblasts create the nano-composite structure of bone by secreting a collagenous extracellular matrix (ECM) on which apatite crystals subsequently form. However, despite their requisite function in building bone and decades of observations describing intracellular calcium phosphate, the precise role osteoblasts play in mediating bone apatite formation remains largely unknown. To better understand the relationship between intracellular and extracellular mineralization, we combined a sample-preparation method that simultaneously preserved mineral, ions, and ECM with nano-analytical electron microscopy techniques to examine osteoblasts in an in vitro model of bone formation. We identified calcium phosphate both within osteoblast mitochondrial granules and intracellular vesicles that transported material to the ECM. Moreover, we observed calcium-containing vesicles conjoining mitochondria, which also contained calcium, suggesting a storage and transport mechanism. Our observations further highlight the important relationship between intracellular calcium phosphate in osteoblasts and their role in mineralizing the ECM. These observations may have important implications in deciphering both how normal bone forms and in understanding pathological mineralization.
Four crystal structures are presented of histone deacetylase 6 (HDAC6) complexes with para-substituted phenylhydromaxamate inhibitors, including bulky peptoids. These structures provide insight ...regarding the design of capping groups that confer selectivity for binding to HDAC6, specifically with regard to interactions in a pocket formed by the L1 loop. Capping group interactions may also influence hydroxamate-Zn2+ coordination with monodentate or bidentate geometry.
The phenothiazine system was identified as a favorable cap group for potent and selective histone deacetylase 6 (HDAC6) inhibitors. Here, we report the preparation and systematic variation of ...phenothiazines and their analogues containing a benzhydroxamic acid moiety as the zinc-binding group. We evaluated their ability to selectively inhibit HDAC6 by a recombinant HDAC enzyme assay, by determining the protein acetylation levels in cells by western blotting (tubulin vs histone acetylation), and by assessing their effects on various cancer cell lines. Structure–activity relationship studies revealed that incorporation of a nitrogen atom into the phenothiazine framework results in increased potency and selectivity for HDAC6 (more than 500-fold selectivity relative to the inhibition of HDAC1, HDAC4, and HDAC8), as rationalized by molecular modeling and docking studies. The binding mode was confirmed by co-crystallization of the potent azaphenothiazine inhibitor with catalytic domain 2 from Danio rerio HDAC6.
Nicotinamide adenine dinucleotide (NAD⁺) is a critical coenzyme for cellular energy metabolism. The aim of the present study was to determine the importance of brown and white adipose tissue (BAT and ...WAT) NAD⁺ metabolism in regulating whole-body thermogenesis and energy metabolism. Accordingly, we generated and analyzed adipocyte-specific nicotinamide phosphoribosyltransferase (Nampt) knockout (ANKO) and brown adipocyte-specific Nampt knockout (BANKO) mice because NAMPT is the rate-limiting NAD⁺ biosynthetic enzyme. We found ANKO mice, which lack NAMPT in both BAT andWAT, had impaired gene programs involved in thermogenesis and mitochondrial function in BAT and a blunted thermogenic (rectal temperature, BAT temperature, and whole-body oxygen consumption) response to acute cold exposure, prolonged fasting, and administration of β-adrenergic agonists (norepinephrine and CL-316243). In addition, the absence of NAMPT in WAT markedly reduced adrenergic-mediated lipolytic activity, likely through inactivation of the NAD⁺–SIRT1–caveolin-1 axis, which limits an important fuel source fatty acid for BAT thermogenesis. These metabolic abnormalities were rescued by treatment with nicotinamide mononucleotide (NMN), which bypasses the block in NAD⁺ synthesis induced by NAMPT deficiency. Although BANKO mice, which lack NAMPT in BAT only, had BAT cellular alterations similar to the ANKO mice, BANKO mice had normal thermogenic and lipolytic responses. We also found NAMPT expression in supraclavicular adipose tissue (where human BAT is localized) obtained from human subjects increased during cold exposure, suggesting our finding in rodents could apply to people. These results demonstrate that adipose NAMPT-mediated NAD⁺ biosynthesis is essential for regulating adaptive thermogenesis, lipolysis, and whole-body energy metabolism.
Spatial and temporal patterns of rainfall are governed by complex interactions between climate and landscape perturbations including deforestation, fire, and drought. Previous research demonstrated ...that rainfall in portions of the Amazon Basin has intensified, resulting in more extreme droughts and floods. The basin has global impacts on climate and hydrologic cycles; thus, it is critical to understand how precipitation patterns and intensity are changing. Due to insufficient precipitation gauges, we analyzed the variability and seasonality of rainfall over the Amazon Basin from 1982 to 2018 using high-resolution gridded precipitation products. We developed several precipitation indices and analyzed their trends using the Mann–Kendall test (Mann
1945
; Kendall, 1975) to identify significant changes in rainfall patterns over time and space. Our results show landscape scale changes in the timing and intensity of rainfall events. Specifically, wet areas of the western Basin have become significantly wetter since 1982, with an increase of 182 mm of rainfall per year. In the eastern and southern regions, where deforestation is widespread, a significant drying trend is evident. Additionally, local alterations to precipitation patterns were also observed. For example, the Tocantins region has had a significant increase in the number of dry days during both wet and dry seasons, increasing by about 1 day per year. Surprisingly, changes in rainfall amount and number of dry days do not consistently align. Broadly, over this 37-year period, wet areas are trending wetter and dry areas are trending drier, while spatial anomalies show structure at the scale of hundreds of kilometers.
Trapoxin A is a microbial cyclic tetrapeptide that is an essentially irreversible inhibitor of class I histone deacetylases (HDACs). The inhibitory warhead is the α,β-epoxyketone side-chain of ...(2S,9S)-2-amino-8-oxo-9,10-epoxydecanoic acid (l-Aoe), which mimics the side-chain of the HDAC substrate acetyl-l-lysine. We now report the crystal structure of the HDAC8–trapoxin A complex at 1.24 Å resolution, revealing that the ketone moiety of l-Aoe undergoes nucleophilic attack to form a zinc-bound tetrahedral gem-diolate that mimics the tetrahedral intermediate and its flanking transition states in catalysis. Mass spectrometry, activity measurements, and isothermal titration calorimetry confirm that trapoxin A binds tightly (K d = 3 ± 1 nM) and does not covalently modify the enzyme, so the epoxide moiety of l-Aoe remains intact. Comparison of the HDAC8–trapoxin A complex with the HDAC6-HC toxin complex provides new insight regarding the inhibitory potency of l-Aoe-containing natural products against class I and class II HDACs.
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