Architecture and regulation of filamentous human cystathionine beta-synthase.

Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.

High-Throughput Screening Reveals New Glutaminase Inhibitor Molecules.

The glutaminase (GLS) enzyme hydrolyzes glutamine into glutamate, an important anaplerotic source for the tricarboxylic acid cycle in rapidly growing cancer cells under the Warburg effect. Glutamine-derived α-ketoglutarate is also an important cofactor of chromatin-modifying enzymes, and through epigenetic changes, it keeps cancer cells in an undifferentiated state. Moreover, glutamate is an important neurotransmitter, and deregulated glutaminase activity in the nervous system underlies several neurological disorders. Given the proven importance of glutaminase for critical diseases, we describe the development of a new coupled enzyme-based fluorescent glutaminase activity assay formatted for 384-well plates for high-throughput screening (HTS) of glutaminase inhibitors. We applied the new methodology to screen a ∼30,000-compound library to search for GLS inhibitors. The HTS assay identified 11 glutaminase inhibitors as hits that were characterized by in silico, biochemical, and glutaminase-based cellular assays. A structure-activity relationship study on the most promising hit (C9) allowed the discovery of a derivative, C9.22, with enhanced in vitro and cellular glutaminase-inhibiting activity. In summary, we discovered a new glutaminase inhibitor with an innovative structural scaffold and described the molecular determinants of its activity.

Several Faces of Glutaminase Regulation in Cells.

The cancer target glutaminase (GLS) has proven to be a fascinating protein. Since it was first described to be regulated by the oncogene Myc 10 years ago, several other transcriptional, posttranscriptional, and posttranslational regulatory mechanisms have emerged, and the list is growing. A recent study by Deng and colleagues revealed that an antisense (AS) long noncoding RNA named GLS-AS, which is negatively regulated by Myc, downregulates GLS in pancreatic cancer. The Myc/GLS-AS/GLS regulatory axis is activated by nutrient stress, which is important for the often hypovascular pancreatic cancer, displaying the significance of GLS for the progression of this highly lethal type of cancer.See related article by Deng et al., p. 1398.

Using microRNA Networks to Understand Cancer.

Human cancers are characterized by deregulated expression of multiple microRNAs (miRNAs), involved in essential pathways that confer the malignant cells their tumorigenic potential. Each miRNA can regulate hundreds of messenger RNAs (mRNAs), while various miRNAs can control the same mRNA. Additionally, many miRNAs regulate and are regulated by other species of non-coding RNAs, such as circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs). For this reason, it is extremely difficult to predict, study, and analyze the precise role of a single miRNA involved in human cancer, considering the complexity of its connections. Focusing on a single miRNA molecule represents a limited approach. Additional information could come from network analysis, which has become a common tool in the biological field to better understand molecular interactions. In this review, we focus on the main types of networks (monopartite, association networks and bipartite) used for analyzing biological data related to miRNA function. We briefly present the important steps to take when generating networks, illustrating the theory with published examples and with future perspectives of how this approach can help to better select miRNAs that can be therapeutically targeted in cancer.

Design and synthesis of cenocladamide analogues and their evaluation against breast cancer cell lines.

This work describes the total synthesis of the alkaloid cenocladamide and a concise library of nine structural analogues aiming at their evaluation against the breast cancer cell line MDA-MB-231. The most promising compound (3; IC50 = 6.6 µM) was also evaluated in a panel of seven breast cancer cell lines and two non-tumorigenic cell lines. We further conducted an initial investigation on the mechanism of action of analogue 3, which lacks the endocyclic double bond when compared to cenocladamide. The present study presents the discovery of a cenocladamide analogue with interesting cytotoxic activity, which could be useful for further optimization towards new chemotherapeutic agents for breast cancer treatment.

Unsaturated fatty acids as high-affinity ligands of the C-terminal Per-ARNT-Sim domain from the Hypoxia-inducible factor 3α.

Hypoxia-inducible transcription factors (HIF) form heterodimeric complexes that mediate cell responses to hypoxia. The oxygen-dependent stability and activity of the HIF-α subunits is traditionally associated to post-translational modifications such as hydroxylation, acetylation, ubiquitination, and phosphorylation. Here we report novel evidence showing that unsaturated fatty acids are naturally occurring, non-covalent structural ligands of HIF-3α, thus providing the initial framework for exploring its exceptional role as a lipid sensor under hypoxia.

Functional diversification of cerato-platanins in Moniliophthora perniciosa as seen by differential expression and protein function specialization.

Cerato-platanins (CP) are small, cysteine-rich fungal-secreted proteins involved in the various stages of the host-fungus interaction process, acting as phytotoxins, elicitors, and allergens. We identified 12 CP genes (MpCP1 to MpCP12) in the genome of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao, and showed that they present distinct expression profiles throughout fungal development and infection. We determined the X-ray crystal structures of MpCP1, MpCP2, MpCP3, and MpCP5, representative of different branches of a phylogenetic tree and expressed at different stages of the disease. Structure-based biochemistry, in combination with nuclear magnetic resonance and mass spectrometry, allowed us to define specialized capabilities regarding self-assembling and the direct binding to chitin and N-acetylglucosamine (NAG) tetramers, a fungal cell wall building block, and to map a previously unknown binding region in MpCP5. Moreover, fibers of MpCP2 were shown to act as expansin and facilitate basidiospore germination whereas soluble MpCP5 blocked NAG6-induced defense response. The correlation between these roles, the fungus life cycle, and its tug-of-war interaction with cacao plants is discussed.

Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism.

Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.

Targeting mitochondrial glutaminase activity inhibits oncogenic transformation.

Rho GTPases impact a number of activities important for oncogenesis. We describe a small molecule inhibitor that blocks oncogenic transformation induced by various Rho GTPases in fibroblasts, and the growth of human breast cancer and B lymphoma cells, without affecting normal cells. We identify the target of this inhibitor to be the metabolic enzyme glutaminase, which catalyzes the hydrolysis of glutamine to glutamate. We show that transformed fibroblasts and breast cancer cells exhibit elevated glutaminase activity that is dependent on Rho GTPases and NF-κB activity, and is blocked by the small molecule inhibitor. These findings highlight a previously unappreciated connection between Rho GTPase activation and cellular metabolism and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.

The molecular basis for the regulation of the cap-binding complex by the importins.

The binding of capped RNAs to the cap-binding complex (CBC) in the nucleus, and their dissociation from the CBC in the cytosol, represent essential steps in RNA processing. Here we show how the nucleocytoplasmic transport proteins importin-alpha and importin-beta have key roles in regulating these events. As a first step toward understanding the molecular basis for this regulation, we determined a 2.2-A resolution X-ray structure for a CBC-importin-alpha complex that provides a detailed picture for how importin-alpha binds to the CBP80 subunit of the CBC. Through a combination of biochemical studies, X-ray crystallographic information and small-angle scattering experiments, we then determined how importin-beta binds to the CBC through its CBP20 subunit. Together, these studies enable us to propose a model describing how importin-beta stimulates the dissociation of capped RNA from the CBC in the cytosol following its nuclear export.

X-ray crystal structures reveal two activated states for RhoC.

RhoC is a member of the Rho family of Ras-related (small) GTPases and shares significant sequence similarity with the founding member of the family, RhoA. However, despite their similarity, RhoA and RhoC exhibit different binding preferences for effector proteins and give rise to distinct cellular outcomes, with RhoC being directly implicated in the invasiveness of cancer cells and the development of metastasis. While the structural analyses of the signaling-active and -inactive states of RhoA have been performed, thus far, the work on RhoC has been limited to an X-ray structure for its complex with the effector protein, mDia1 (for mammalian Diaphanous 1). Therefore, in order to gain insights into the molecular basis for RhoC activation, as well as clues regarding how it mediates distinct cellular responses relative to those induced by RhoA, we have undertaken a structural comparison of RhoC in its GDP-bound (signaling-inactive) state versus its GTP-bound (signaling-active) state as induced by the nonhydrolyzable GTP analogues, guanosine 5'-(beta,gamma-iminotriphosphate) (GppNHp) and guanosine 5'-(3-O-thiotriphosphate) (GTPgammaS). Interestingly, we find that GppNHp-bound RhoC only shows differences in its switch II domain, relative to GDP-bound RhoC, whereas GTPgammaS-bound RhoC exhibits differences in both its switch I and switch II domains. Given that each of the nonhydrolyzable GTP analogues is able to promote the binding of RhoC to effector proteins, these results suggest that RhoC can undergo at least two conformational transitions during its conversion from a signaling-inactive to a signaling-active state, similar to what has recently been proposed for the H-Ras and M-Ras proteins. In contrast, the available X-ray structures for RhoA suggest that it undergoes only a single conformational transition to a signaling-active state. These and other differences regarding the changes in the switch domains accompanying the activation of RhoA and RhoC provide plausible explanations for the functional specificity exhibited by the two GTPases.

Ajulemic acid, a synthetic nonpsychoactive cannabinoid acid, bound to the ligand binding domain of the human peroxisome proliferator-activated receptor gamma.

Ajulemic acid (AJA) is a synthetic analog of THC-11-oic acid, a metabolite of tetrahydrocannabinol (THC), the major active ingredient of the recreational drug marijuana derived from the plant Cannabis sativa. AJA has potent analgesic and anti-inflammatory activity in vivo, but without the psychotropic action of THC. However, its precise mechanism of action remains unknown. Biochemical studies indicate that AJA binds directly and selectively to the isotype gamma of the peroxisome proliferator-activated receptor (PPARgamma) suggesting that this may be a pharmacologically relevant receptor for this compound and a potential target for drug development in the treatment of pain and inflammation. Here, we report the crystal structure of the ligand binding domain of the gamma isotype of human PPAR in complex with ajulemic acid, determined at 2.8-A resolution. Our results show a binding mode that is compatible with other known partial agonists of PPAR, explaining their moderate activation of the receptor, as well as the structural basis for isotype selectivity, as observed previously in vitro. The structure also provides clues to the understanding of partial agonism itself, suggesting a rational approach to the design of molecules capable of activating the receptor at levels that avoid undesirable side effects.

Human thyroid receptor forms tetramers in solution, which dissociate into dimers upon ligand binding.

Thyroid hormone nuclear receptors (TRs) bind to DNA and activate transcription as heterodimers with the retinoid X receptor (RXR) or as homodimers or monomers. RXR also binds to DNA and activates transcription as homodimers but can, in addition, self-associate into homotetramers in the absence of ligand and DNA templates. It is thought that homotetramer formation serves to sequester excess RXRs into an inactive pool within the cell. Here, we report systematic studies of the multimeric state of a recombinant human TRbeta1 truncation (hTRbeta1deltaAB) that encompasses the complete DNA binding domain and ligand binding domain in solution. Native gel electrophoresis, chemical crosslinking, gel filtration, and dynamic light scattering experiments reveal that hTRbeta1deltaAB forms a mixture of monomers, dimers, and tetramers. Like RXR, increasing protein concentration shifts the equilibrium between TR multimers toward tetramer formation, whereas binding of cognate thyroid hormone leads to dissociation of tetramers and increased formation of dimers. This work represents the first evidence that apo-hTRbeta1 forms homotetramers. The findings raise the possibility that tetramer formation provides an additional, and previously unsuspected, level of control of TR activity and that the capacity for homotetramer formation may be more widespread in the nuclear receptor family than previously thought.

[Molecular mechanism of thyroid hormone action]. / Mecanismo molecular da ação do hormônio tireoideano.

Thyroid hormones (TH) are involved in normal differentiation, growth, and metabolism in several tissues of all vertebrates. Their actions are mediated by the TH receptors (TRs), members of the nuclear hormone receptor superfamily. These receptors are transcription factors that bind to DNA on specific sequences, the TR response element (TREs), in promoters of target genes. Two genes encode TRs, alpha e beta, located in chromosomes 17 and 3, respectively. These isoforms show different functions and exhibit a tissue specific expression. TRs function as monomers, homodimers or heterodimers with retinoid X receptor (RXR) and modulate transcription activity (repression or activation) by interacting with co-repressor and co-activators, which associate with TR in the absence or presence of T3, respectively. Understanding the molecular mechanism of TR action and the definition of its crystallographic structure will provide new insights into transcription mechanisms and will facilitate the design of new drugs with greater therapeutic value.

Mecanismo molecular da ação do hormônio tireoideano / Molecular mechanism of thyroid hormone action

Os hormônios tireoideanos (HTs) são necessários para a diferenciação, crescimento e metabolismo de diversos tecidos de vertebrados. Seus efeitos são mediados pelos receptores do hormônio tireoideano (TRs), membros da superfamília dos receptores nucleares. Estes receptores são fatores de transcrição modulares que se ligam em seqüências específicas do DNA denominadas elementos responsivos ao TR, que são encontrados nos promotores dos genes regulados pelo HT. Os TRs são codificados por dois genes distintos, alfae beta, localizados nos cromossomos 17 e 3, respectivamente. Estas isoformas apresentam diferentes funções e sua expressão é específica para cada tecido. O TR se liga ao DNA como monômero, homodímero ou heterodímero com o receptor de retinóide X (RXR). Além disso, o TR modula a atividade transcricional (repressão ou ativação) através da interação com correpressores e co-ativadores, na ausência e na presença do T3, respectivamente. A compreensão do mecanismo molecular da ação do receptor do hormônio tireoideano e a definição de sua estrutura cristalográfica contribuirão para a aquisição de novos conceitos envolvidos na transcrição e nos distúrbios hormonais presentes nas doenças endócrinas, assim como facilitará o desenho de novas drogas, agonistas ou antagonistas, com grande valor terapêutico.

Low resolution structures of the retinoid X receptor DNA-binding and ligand-binding domains revealed by synchrotron X-ray solution scattering.

Nuclear receptors are ligand-inducible transcription factors that share structurally related DNA-binding (DBD) and ligand-binding (LBD) domains. Biochemical and structural studies have revealed the modular nature of DBD and LBD. Nevertheless, the domains function in concert in vivo. While high-resolution crystal structures of nuclear receptor DBDs and LBDs are available, there are no x-ray structural studies of nuclear receptor proteins containing multiple domains. We report the solution structures of the human retinoid X receptor DBD-LBD (hRXRalphaDeltaAB) region. We obtained ab initio shapes of hRXRalphaDeltaAB dimer and tetramer to 3.3 and 1.7 nm resolutions, respectively, and established the position and orientation of the DBD and LBD by fitting atomic coordinates of hRXRalpha DBD and LBD. The dimer is U-shaped with DBDs spaced at approximately 2 nm in a head to head orientation forming an angle of about 10 degrees with respect to each other and with an extensive interface area provided by the LBD. The tetramer is a more elongated X-shaped molecule formed by two dimers in head to head arrangement in which the DBDs are extended from the structure and spaced at about 6 nm. The close proximity of DBDs in dimers may facilitate homodimer formation on DNA; however, for the homodimer to bind to a DNA element containing two directly repeated half-sites, one of the DBDs would need to rotate with respect to the other element. By contrast, the separation of DBDs in the tetramers may account for their decreased ability to recognize DNA.