Advances in our structural understanding of orphan nuclear receptors.

Nuclear receptors (NRs) are key players in the regulation of gene expression, coordinating protein assemblies upon their surfaces. NRs are regulated by ligand binding, which remodels the interaction surfaces and subsequently influences macromolecular complex formation. Structural biology has been instrumental in the discovery of some of these ligands, but there are still orphan NRs (ONRs) whose bona fide ligands have yet to be identified. Over the past decade, fundamental structural and functional breakthroughs have led to a deeper understanding of ONR actions and their multidomain organization. Here, we summarize the structural advances in ONRs with implications for the therapeutic treatment of diseases such as metabolic syndrome and cancer.

The 26S proteasome: assembly and function of a destructive machine.

The heart of the ubiquitin-mediated degradation pathway, the 26S proteasome, endoproteolytically cleaves most intracellular proteins, thereby maintaining biological homeostasis and regulating many crucial processes in the cell. This hydrolyzing machine comprises more than 30 different subunits, which perform different functions including the recognition, unfolding, translocating and cleavage of protein substrates. Thus, careful assemblage and regulation of the 26S proteasome is essential to ensure correct positioning and function of each subunit, thereby preserving the delicate cellular balance between protein synthesis and degradation. Here, we review the most current research on the 26S proteasome assembly pathway, and describe the mechanism used by the cell to manage the complex structure and functions of the proteasome.

Omuralide and vibralactone: differences in the proteasome- ß-lactone-γ-lactam binding scaffold alter target preferences.

Despite their structural similarity, the natural products omuralide and vibralactone have different biological targets. While omuralide blocks the chymotryptic activity of the proteasome with an IC50 value of 47 nM, vibralactone does not have any effect at this protease up to a concentration of 1 mM. Activity-based protein profiling in HeLa cells revealed that the major targets of vibralactone are APT1 and APT2.

Structure of the homodimeric androgen receptor ligand-binding domain.

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.

Allosteric mechanisms of nuclear receptors: insights from computational simulations.

The traditional structural view of allostery defines this key regulatory mechanism as the ability of one conformational event (allosteric site) to initiate another in a separate location (active site). In recent years computational simulations conducted to understand how this phenomenon occurs in nuclear receptors (NRs) has gained significant traction. These results have yield insights into allosteric changes and communication mechanisms that underpin ligand binding, coactivator binding site formation, post-translational modifications, and oncogenic mutations. Moreover, substantial efforts have been made in understanding the dynamic processes involved in ligand binding and coregulator recruitment to different NR conformations in order to predict cell/tissue-selective pharmacological outcomes of drugs. They also have improved the accuracy of in silico screening protocols so that nowadays they are becoming part of optimisation protocols for novel therapeutics. Here we summarise the important contributions that computational simulations have made towards understanding the structure/function relationships of NRs and how these can be exploited for rational drug design.

Dimerized linear mimics of a natural cyclopeptide (TMC-95A) are potent noncovalent inhibitors of the eukaryotic 20S proteasome.

Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors used in cancer therapy. Starting from a noncovalent linear mimic of TMC-95A, a series of dimerized inhibitors using polyaminohexanoic acid spacers has been designed and optimized to target simultaneously two of the six active sites of the eukaryotic 20S proteasome. The homodimerized compounds actively inhibited chymotrypsin-like (Ki = 6-11 nM) and trypsin-like activities, whereas postacid activity was poorly modified. The noncovalent binding mode was ascertained by X-ray crystallography of the inhibitors complexed with the yeast 20S proteasome. The inhibition of proteasomal activities in human cells was evaluated. The use of the multivalency inhibitor concept has produced highly efficient and selective noncovalent compounds (no inhibition of calpain and cathepsin) that have potential therapeutic advantages compared to covalent binders such as bortezomib and carfilzomib.

Incorporation of non-natural amino acids improves cell permeability and potency of specific inhibitors of proteasome trypsin-like sites.

Proteasomes degrade the majority of proteins in mammalian cells by a concerted action of three distinct pairs of active sites. The chymotrypsin-like sites are targets of antimyeloma agents bortezomib and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple myeloma cells to these agents. Here we describe systematic effort to develop inhibitors with improved potency and cell permeability, yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone (4a, LU-102), and a fluorescent activity-based probe for this site. X-ray structures of 4a and related inhibitors complexed with yeast proteasomes revealed the structural basis for specificity. Nontoxic to myeloma cells when used as a single agent, 4a sensitized them to bortezomib and carfilzomib. This sensitizing effect was much stronger than the synergistic effects of histone acetylase inhibitors or additive effects of doxorubicin and dexamethasone, raising the possibility that combinations of inhibitors of the trypsin-like site with bortezomib or carfilzomib would have stronger antineoplastic activity than combinations currently used clinically.

Analysing properties of proteasome inhibitors using kinetic and X-ray crystallographic studies.

The combination of X-ray crystallography and kinetic studies of proteasome:ligand complexes has proven to be an important tool in inhibitor analysis of this crucial protein degradation machinery. Here, we describe in detail the purification protocols, proteolytic activity assays, crystallisation methods, and structure determination for the yeast 20S proteasome (CP) in complex with its inhibitors. The fusion of these advanced techniques offers the opportunity to further optimise drugs which are already tested in different clinical phase studies, as well as to design new promising proteasome lead structures which might be suitable for their application in medicine, plant protection, and antibiotics.

Peptido sulfonyl fluorides as new powerful proteasome inhibitors.

A new class of potent proteasome inhibitors is described, of which the members contain an amino acid inspired sulfonyl fluoride as the electrophilic trap. In total, 24 peptido sulfonyl fluoride inhibitors have been designed and synthesized, which were inspired by the backbone sequences of the proteasome inhibitors bortezomib, epoxomicin, and Cbz-Leu(3)-aldehyde. Nine of them were very potent proteasome inhibitors, the best of which had an IC(50) of 7 nM. A number of the peptido sulfonyl fluoride inhibitors were found to be highly selective for the ß5 proteasome subunit.

How ATPases unravel a mystery.

Principles of intracellular protein degradation remain among the most challenging questions in cell biology. Here, we discuss Wang and colleagues' crystal structure elucidation of the intermediate domain of Mpa, a regulatory particle of Mtb proteasome, the core proteolytic machinery of Mycobacterium tuberculosis.