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.

Aster-dependent nonvesicular transport facilitates dietary cholesterol uptake.

Intestinal absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1) assists in the initial step of dietary cholesterol uptake, but how cholesterol moves downstream of NPC1L1 is unknown. We show that Aster-B and Aster-C are critical for nonvesicular cholesterol movement in enterocytes. Loss of NPC1L1 diminishes accessible plasma membrane (PM) cholesterol and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate PM cholesterol and show endoplasmic reticulum cholesterol depletion. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, the Aster pathway can be targeted with a small-molecule inhibitor to manipulate cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption.

Aster-dependent non-vesicular transport facilitates dietary cholesterol uptake.

Intestinal cholesterol absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1), the target of the drug ezetimibe (EZ), assists in the initial step of dietary cholesterol uptake. However, how cholesterol moves downstream of NPC1L1 is unknown. Here we show that Aster-B and Aster-C are critical for non-vesicular cholesterol movement in enterocytes, bridging NPC1L1 at the plasma membrane (PM) and ACAT2 in the endoplasmic reticulum (ER). Loss of NPC1L1 diminishes accessible PM cholesterol in enterocytes and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate cholesterol at the PM and display evidence of ER cholesterol depletion, including decreased cholesterol ester stores and activation of the SREBP-2 transcriptional pathway. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, we show that the Aster pathway can be targeted with a small molecule inhibitor to manipulate dietary cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption. One-Sentence Summary: Identification of a targetable pathway for regulation of dietary cholesterol absorption.

A potential histone-chaperone activity for the MIER1 histone deacetylase complex.

Histone deacetylases 1 and 2 (HDAC1/2) serve as the catalytic subunit of six distinct families of nuclear complexes. These complexes repress gene transcription through removing acetyl groups from lysine residues in histone tails. In addition to the deacetylase subunit, these complexes typically contain transcription factor and/or chromatin binding activities. The MIER:HDAC complex has hitherto been poorly characterized. Here, we show that MIER1 unexpectedly co-purifies with an H2A:H2B histone dimer. We show that MIER1 is also able to bind a complete histone octamer. Intriguingly, we found that a larger MIER1:HDAC1:BAHD1:C1QBP complex additionally co-purifies with an intact nucleosome on which H3K27 is either di- or tri-methylated. Together this suggests that the MIER1 complex acts downstream of PRC2 to expand regions of repressed chromatin and could potentially deposit histone octamer onto nucleosome-depleted regions of DNA.

Histone H2B Deacylation Selectivity: Exploring Chromatin's Dark Matter with an Engineered Sortase.

We describe a new method to produce histone H2B by semisynthesis with an engineered sortase transpeptidase. N-Terminal tail site-specifically modified acetylated, lactylated, and ß-hydroxybutyrylated histone H2Bs were incorporated into nucleosomes and investigated as substrates of histone deacetylase (HDAC) complexes and sirtuins. A wide range of rates and site-specificities were observed by these enzyme forms suggesting distinct biological roles in regulating chromatin structure and epigenetics.

Structure-Guided Approach to Relieving Transcriptional Repression in Resistance to Thyroid Hormone α.

Mutations in thyroid hormone receptor α (TRα), a ligand-inducible transcription factor, cause resistance to thyroid hormone α (RTHα). This disorder is characterized by tissue-specific hormone refractoriness and hypothyroidism due to the inhibition of target gene expression by mutant TRα-corepressor complexes. Using biophysical approaches, we show that RTHα-associated TRα mutants devoid of ligand-dependent transcription activation function unexpectedly retain the ability to bind thyroid hormone. Visualization of the ligand T3 within the crystal structure of a prototypic TRα mutant validates this notion. This finding prompted the synthesis of different thyroid hormone analogues, identifying a lead compound, ES08, which dissociates corepressor from mutant human TRα more efficaciously than T3. ES08 rescues developmental anomalies in a zebrafish model of RTHα and induces target gene expression in TRα mutation-containing cells from an RTHα patient more effectively than T3. Our observations provide proof of principle for developing synthetic ligands that can relieve transcriptional repression by the mutant TRα-corepressor complex for treatment of RTHα.

Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

The topology of chromatin-binding domains in the NuRD deacetylase complex.

Class I histone deacetylase complexes play essential roles in many nuclear processes. Whilst they contain a common catalytic subunit, they have diverse modes of action determined by associated factors in the distinct complexes. The deacetylase module from the NuRD complex contains three protein domains that control the recruitment of chromatin to the deacetylase enzyme, HDAC1/2. Using biochemical approaches and cryo-electron microscopy, we have determined how three chromatin-binding domains (MTA1-BAH, MBD2/3 and RBBP4/7) are assembled in relation to the core complex so as to facilitate interaction of the complex with the genome. We observe a striking arrangement of the BAH domains suggesting a potential mechanism for binding to di-nucleosomes. We also find that the WD40 domains from RBBP4 are linked to the core with surprising flexibility that is likely important for chromatin engagement. A single MBD2 protein binds asymmetrically to the dimerisation interface of the complex. This symmetry mismatch explains the stoichiometry of the complex. Finally, our structures suggest how the holo-NuRD might assemble on a di-nucleosome substrate.

Hyperthyroxinemia and Hypercortisolemia due to Familial Dysalbuminemia.

A 23-year-old man and his grandmother with hyperthyroxinemia and hypercortisolemia were heterozygous for an ALB mutation (p. Arg218Pro), known to cause familial dysalbuminemic hyperthyroxinemia (FDH). However, serum-free cortisol levels in these individuals were normal and total cortisol concentrations fell markedly after depletion of albumin from their serum. We conclude that binding of steroid as well as iodothyronines to mutant albumin causes raised circulating cortisol as well as thyroid hormones in euthyroid euadrenal individuals with R218P FDH, with potential for misdiagnosis, unnecessary investigation, and inappropriate treatment.

The MiDAC histone deacetylase complex is essential for embryonic development and has a unique multivalent structure.

MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function.

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.

Mechanism of Crosstalk between the LSD1 Demethylase and HDAC1 Deacetylase in the CoREST Complex.

The transcriptional corepressor complex CoREST is one of seven histone deacetylase complexes that regulate the genome through controlling chromatin acetylation. The CoREST complex is unique in containing both histone demethylase and deacetylase enzymes, LSD1 and HDAC1, held together by the RCOR1 scaffold protein. To date, it has been assumed that the enzymes function independently within the complex. Now, we report the assembly of the ternary complex. Using both structural and functional studies, we show that the activity of the two enzymes is closely coupled and that the complex can exist in at least two distinct states with different kinetics. Electron microscopy of the complex reveals a bi-lobed structure with LSD1 and HDAC1 enzymes at opposite ends of the complex. The structure of CoREST in complex with a nucleosome reveals a mode of chromatin engagement that contrasts with previous models.

Heme binding to human CLOCK affects interactions with the E-box.

The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.

Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells.

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

A Pharmacogenetic Approach to the Treatment of Patients With PPARG Mutations.

Loss-of-function mutations in PPARG cause familial partial lipodystrophy type 3 (FPLD3) and severe metabolic disease in many patients. Missense mutations in PPARG are present in ∼1 in 500 people. Although mutations are often binarily classified as benign or deleterious, prospective functional classification of all missense PPARG variants suggests that their impact is graded. Furthermore, in testing novel mutations with both prototypic endogenous (e.g., prostaglandin J2 [PGJ2]) and synthetic ligands (thiazolidinediones, tyrosine agonists), we observed that synthetic agonists selectively rescue function of some peroxisome proliferator-activated receptor-γ (PPARγ) mutants. We report on patients with FPLD3 who harbor two such PPARγ mutations (R308P and A261E). Both PPARγ mutants exhibit negligible constitutive or PGJ2-induced transcriptional activity but respond readily to synthetic agonists in vitro, with structural modeling providing a basis for such differential ligand-dependent responsiveness. Concordant with this finding, dramatic clinical improvement was seen after pioglitazone treatment of a patient with R308P mutant PPARγ. A patient with A261E mutant PPARγ also responded beneficially to rosiglitazone, although cardiomyopathy precluded prolonged thiazolidinedione use. These observations indicate that detailed structural and functional classification can be used to inform therapeutic decisions in patients with PPARG mutations.

Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors.

Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin's favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.

Expression and Purification of Protein Complexes Suitable for Structural Studies Using Mammalian HEK 293F Cells.

Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not produce significant yields of soluble protein. This may result from failure to efficiently transcribe/translate the required protein or may result from the formation of insoluble aggregates known as inclusion bodies. Mammalian expression systems can often produce natively folded proteins, sometimes with native post-translational modifications. However, such expression systems are underutilized due to the perception that they are costly, technically challenging and result in limited protein yields. In fact, HEK 293F cells are straightforward to grow, transfect with high efficiency and often produce significant yields of recombinant proteins. In this unit, we describe a method to express and purify milligram quantities of a human protein complex from HEK 293F cells grown in suspension transiently transfected with the appropriate plasmids. © 2017 by John Wiley & Sons, Inc.

Contrasting Phenotypes in Resistance to Thyroid Hormone Alpha Correlate with Divergent Properties of Thyroid Hormone Receptor α1 Mutant Proteins.

BACKGROUND: Resistance to thyroid hormone alpha (RTHα), a disorder characterized by tissue-selective hypothyroidism and near-normal thyroid function tests due to thyroid receptor alpha gene mutations, is rare but probably under-recognized. This study sought to correlate the clinical characteristics and response to thyroxine (T4) therapy in two adolescent RTHα patients with the properties of the THRA mutation, affecting both TRα1 and TRα2 proteins, they harbored. METHODS: Clinical, auxological, biochemical, and physiological parameters were assessed in each patient at baseline and after T4 therapy. RESULTS: Heterozygous THRA mutations occurring de novo were identified in a 17-year-old male (patient P1; c.788C>T, p.A263V mutation) investigated for mild pubertal delay and in a 15-year-old male (patient P2; c.821T>C, p.L274P mutation) with short stature (0.4th centile), skeletal dysplasia, dysmorphic facies, and global developmental delay. Both individuals exhibited macrocephaly, delayed dentition, and constipation, together with a subnormal T4/triiodothyronine (T3) ratio, low reverse T3 levels, and mild anemia. When studied in vitro, A263V mutant TRα1 was transcriptionally impaired and inhibited the function of its wild-type counterpart at low (0.01-10 nM) T3 levels, with higher T3 concentrations (100 nM-1 µM) reversing dysfunction and such dominant negative inhibition. In contrast, L274P mutant TRα1 was transcriptionally inert, exerting significant dominant negative activity, only overcome with 10 µM of T3. Mirroring this, normal expression of KLF9, a TH-responsive target gene, was achieved in A263V mutation-containing peripheral blood mononuclear cells following 1 µM of T3 exposure, but with markedly reduced expression levels in L274P mutation-containing peripheral blood mononuclear cells, even with 10 µM of T3. Following T4 therapy, growth, body composition, dyspraxia, and constipation improved in P1, whereas growth retardation and constipation in P2 were unchanged. Neither A263V nor L274P mutations exhibited gain or loss of function in the TRα2 background, and no additional phenotype attributable to this was discerned. CONCLUSIONS: This study correlates a milder clinical phenotype and favorable response to T4 therapy in a RTHα patient (P1) with heterozygosity for mutant TRα1 exhibiting partial, T3-reversible, loss of function. In contrast, a more severe clinical phenotype refractory to hormone therapy was evident in another case (P2) associated with severe, virtually irreversible, dysfunction of mutant TRα1.

Genetic disorders of nuclear receptors.

Following the first isolation of nuclear receptor (NR) genes, genetic disorders caused by NR gene mutations were initially discovered by a candidate gene approach based on their known roles in endocrine pathways and physiologic processes. Subsequently, the identification of disorders has been informed by phenotypes associated with gene disruption in animal models or by genetic linkage studies. More recently, whole exome sequencing has associated pathogenic genetic variants with unexpected, often multisystem, human phenotypes. To date, defects in 20 of 48 human NR genes have been associated with human disorders, with different mutations mediating phenotypes of varying severity or several distinct conditions being associated with different changes in the same gene. Studies of individuals with deleterious genetic variants can elucidate novel roles of human NRs, validating them as targets for drug development or providing new insights into structure-function relationships. Importantly, human genetic discoveries enable definitive disease diagnosis and can provide opportunities to therapeutically manage affected individuals. Here we review germline changes in human NR genes associated with "monogenic" conditions, including a discussion of the structural basis of mutations that cause distinctive changes in NR function and the molecular mechanisms mediating pathogenesis.

Targeting Class I Histone Deacetylases in a "Complex" Environment.

Histone deacetylase (HDAC) inhibitors are proven anticancer therapeutics and have potential in the treatment of many other diseases including HIV infection, Alzheimer's disease, and Friedreich's ataxia. A problem with the currently available HDAC inhibitors is that they have limited specificity and target multiple deacetylases. Designing isoform-selective inhibitors has proven challenging due to similarities in the structure and chemistry of HDAC active sites. However, the fact that HDACs 1, 2, and 3 are recruited to several large multi-subunit complexes, each with particular biological functions, raises the possibility of specifically inhibiting individual complexes. This may be assisted by recent structural and functional information about the assembly of these complexes. Here, we review the available structural information and discuss potential targeting strategies.