Aster-B-dependent estradiol synthesis protects female mice from diet-induced obesity.

Aster proteins mediate the nonvesicular transport of cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER). However, the importance of nonvesicular sterol movement for physiology and pathophysiology in various tissues is incompletely understood. Here we show that loss of Aster-B leads to diet-induced obesity in female but not in male mice, and that this sex difference is abolished by ovariectomy. We further demonstrate that Aster-B deficiency impairs nonvesicular cholesterol transport from the PM to the ER in ovaries in vivo, leading to hypogonadism and reduced estradiol synthesis. Female Aster-B-deficient mice exhibit reduced locomotor activity and energy expenditure, consistent with established effects of estrogens on systemic metabolism. Administration of exogenous estradiol ameliorates the diet-induced obesity phenotype of Aster-B-deficient female mice. These findings highlight the key role of Aster-B-dependent nonvesicular cholesterol transport in regulating estradiol production and protecting females from obesity.

Structural insights into the complex of oncogenic KRas4BG12V and Rgl2, a RalA/B activator.

About a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA, and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human KRas4B and the Ras association (RA) domain of Rgl2 (Rgl2RA), one of the RA-containing RalGEFs. We show that the G12V oncogenic KRas4B mutation changes the interaction kinetics with Rgl2RA The crystal structure of the KRas4BG12V: Rgl2RA complex shows a 2:2 heterotetramer where the switch I and switch II regions of each KRasG12V interact with both Rgl2RA molecules. This structural arrangement is highly similar to the HRasE31K:RALGDSRA crystal structure and is distinct from the well-characterised Ras:Raf complex. Interestingly, the G12V mutation was found at the dimer interface of KRas4BG12V with its partner. Our study reveals a potentially distinct mode of Ras:effector complex formation by RalGEFs and offers a possible mechanistic explanation for how the oncogenic KRas4BG12V hyperactivates the RalA/B pathway.

Diverse nucleosome Site-Selectivity among histone deacetylase complexes.

Histone acetylation regulates chromatin structure and gene expression and is removed by histone deacetylases (HDACs). HDACs are commonly found in various protein complexes to confer distinct cellular functions, but how the multi-subunit complexes influence deacetylase activities and site-selectivities in chromatin is poorly understood. Previously we reported the results of studies on the HDAC1 containing CoREST complex and acetylated nucleosome substrates which revealed a notable preference for deacetylation of histone H3 acetyl-Lys9 vs. acetyl-Lys14 (Wu et al, 2018). Here we analyze the enzymatic properties of five class I HDAC complexes: CoREST, NuRD, Sin3B, MiDAC and SMRT with site-specific acetylated nucleosome substrates. Our results demonstrate that these HDAC complexes show a wide variety of deacetylase rates in a site-selective manner. A Gly13 in the histone H3 tail is responsible for a sharp reduction in deacetylase activity of the CoREST complex for H3K14ac. These studies provide a framework for connecting enzymatic and biological functions of specific HDAC complexes.

Lysine-14 acetylation of histone H3 in chromatin confers resistance to the deacetylase and demethylase activities of an epigenetic silencing complex.

The core CoREST complex (LHC) contains histone deacetylase HDAC1 and histone demethylase LSD1 held together by the scaffold protein CoREST. Here, we analyze the purified LHC with modified peptide and reconstituted semisynthetic mononucleosome substrates. LHC demethylase activity toward methyl-Lys4 in histone H3 is strongly inhibited by H3 Lys14 acetylation, and this appears to be an intrinsic property of the LSD1 subunit. Moreover, the deacetylase selectivity of LHC unexpectedly shows a marked preference for H3 acetyl-Lys9 versus acetyl-Lys14 in nucleosome substrates but this selectivity is lost with isolated acetyl-Lys H3 protein. This diminished activity of LHC to Lys-14 deacetylation in nucleosomes is not merely due to steric accessibility based on the pattern of sensitivity of the LHC enzymatic complex to hydroxamic acid-mediated inhibition. Overall, these studies have revealed how a single Lys modification can confer a composite of resistance in chromatin to a key epigenetic enzyme complex involved in gene silencing.