The evolution and polymorphism of mono-amino acid repeats in androgen receptor and their regulatory role in health and disease.

Androgen receptor (AR) is a key member of nuclear hormone receptors with the longest intrinsically disordered N-terminal domain (NTD) in its protein family. There are four mono-amino acid repeats (polyQ1, polyQ2, polyG, and polyP) located within its NTD, of which two are polymorphic (polyQ1 and polyG). The length of both polymorphic repeats shows clinically important correlations with disease, especially with cancer and neurodegenerative diseases, as shorter and longer alleles exhibit significant differences in expression, activity and solubility. Importantly, AR has also been shown to undergo condensation in the nucleus by liquid-liquid phase separation, a process highly sensitive to protein solubility and concentration. Nonetheless, in prostate cancer cells, AR variants also partition into transcriptional condensates, which have been shown to alter the expression of target gene products. In this review, we summarize current knowledge on the link between AR repeat polymorphisms and cancer types, including mechanistic explanations and models comprising the relationship between condensate formation, polyQ1 length and transcriptional activity. Moreover, we outline the evolutionary paths of these recently evolved amino acid repeats across mammalian species, and discuss new research directions with potential breakthroughs and controversies in the literature.

A Novel Tandem-Tag Purification Strategy for Challenging Disordered Proteins.

Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor.

F/YGG-motif is an intrinsically disordered nucleic-acid binding motif.

Heterogeneous nuclear ribonucleoproteins (hnRNP) function in RNA processing, have RNA-recognition motifs (RRMs) and intrinsically disordered, low-complexity domains (LCDs). While RRMs are drivers of RNA binding, there is only limited knowledge about the RNA interaction by the LCD of some hnRNPs. Here, we show that the LCD of hnRNPA2 interacts with RNA via an embedded Tyr/Gly-rich region which is a disordered RNA-binding motif. RNA binding is maintained upon mutating tyrosine residues to phenylalanines, but abrogated by mutating to alanines, thus we term the RNA-binding region 'F/YGG motif'. The F/YGG motif can bind a broad range of structured (e.g. tRNA) and disordered (e.g. polyA) RNAs, but not rRNA. As the F/YGG otif can also interact with DNA, we consider it a general nucleic acid-binding motif. hnRNPA2 LCD can form dense droplets, by liquid-liquid phase separation (LLPS). Their formation is inhibited by RNA binding, which is mitigated by salt and 1,6-hexanediol, suggesting that both electrostatic and hydrophobic interactions feature in the F/YGG motif. The D290V mutant also binds RNA, which interferes with both LLPS and aggregation thereof. We found homologous regions in a broad range of RNA- and DNA-binding proteins in the human proteome, suggesting that the F/YGG motif is a general nucleic acid-interaction motif.

DisProt in 2022: improved quality and accessibility of protein intrinsic disorder annotation.

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.

DNA-binding domain as the minimal region driving RNA-dependent liquid-liquid phase separation of androgen receptor.

Androgen receptor (AR) is a nuclear hormone receptor that regulates the transcription of genes involved in the development of testis, prostate and the nervous system. Misregulation of AR is a major driver of prostate cancer (PC). The primary agonist of full-length AR is testosterone, whereas its splice variants, for example, AR-v7 implicated in cancer may lack a ligand-binding domain and are thus devoid of proper hormonal control. Recently, it was demonstrated that full-length AR, but not AR-v7, can undergo liquid-liquid phase separation (LLPS) in a cellular model of PC. In a detailed bioinformatics and deletion analysis, we have analyzed which AR region is responsible for LLPS. We found that its DNA-binding domain (DBD) can bind RNA and can undergo RNA-dependent LLPS. RNA regulates its LLPS in a reentrant manner, that is, it has an inhibitory effect at higher concentrations. As RNA binds DBD more weakly than DNA, while both RNA and DNA localizes into AR droplets, its LLPS depends on the relative concentration of the two nucleic acids. The region immediately preceding DBD has no effect on the LLPS propensity of AR, whereas the functional part of its long N-terminal disordered transactivation domain termed activation function 1 (AF1) inhibits AR-v7 phase separation. We suggest that the resulting diminished LLPS tendency of AR-v7 may contribute to the misregulation of the transcription function of AR in prostate cancer.

Targeting an Intrinsically Disordered Protein by Covalent Modification.

Intrinsically disordered proteins (IDPs) play important roles in the regulation of cellular function and in disease, and thus they represent an important group of therapeutic targets. Yet, members of this "disorderome" have not yet been successfully targeted by drugs, primarily because traditional design principles cannot be applied to their highly dynamic, heterogeneous structural states. Binders developed against IDPs so far suffer from very weak binding and inability to act in a cellular context. Here, we describe a possible generic method for the targeting of IDPs via covalent modification, which could entail specific and strong binding and inhibitory potential, making such "warheads" reasonable starting points of drug-development efforts. We demonstrate this principle by addressing the cysteine-specific covalent modification of calpastatin, the IDP inhibitor of the calcium-dependent cysteine protease calpain. We describe the protocol for monitoring the covalent modification of the inhibitor, measuring the Ki of its inhibition and comparing it to the Kd of its interaction with the enzyme. Our premise is that the underlying principles can be easily adapted to screen for molecules targeting other, disease-related, IDPs in the future.

Molecular Motions and Interactions in Aqueous Solutions of Thymosin-ß4 , Stabilin C-Terminal Domain (CTD) and Their 1:1 Complex Studied by 1 H†NMR Spectroscopy.

Wide-line 1 H NMR measurements were extended and all results were reinterpreted in a new thermodynamics-based approach to study aqueous solutions of thymosin-ß4 (Tß4 ), stabilin C-terminal domain (CTD) and their 1:1 complex. The energy distributions of the potential barriers, which control motion of protein-bound water molecules, were determined. Heterogeneous and homogeneous regions were found at the protein-water interface. The measure of heterogeneity gives a quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to 20 % of the whole proteins. About 40 % of the binding sites of free Tß4 become involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tß4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

DisProt 7.0: a major update of the database of disordered proteins.

The Database of Protein Disorder (DisProt, URL: www.disprot.org) has been significantly updated and upgraded since its last major renewal in 2007. The current release holds information on more than 800 entries of IDPs/IDRs, i.e. intrinsically disordered proteins or regions that exist and function without a well-defined three-dimensional structure. We have re-curated previous entries to purge DisProt from conflicting cases, and also upgraded the functional classification scheme to reflect continuous advance in the field in the past 10 years or so. We define IDPs as proteins that are disordered along their entire sequence, i.e. entirely lack structural elements, and IDRs as regions that are at least five consecutive residues without well-defined structure. We base our assessment of disorder strictly on experimental evidence, such as X-ray crystallography and nuclear magnetic resonance (primary techniques) and a broad range of other experimental approaches (secondary techniques). Confident and ambiguous annotations are highlighted separately. DisProt 7.0 presents classified knowledge regarding the experimental characterization and functional annotations of IDPs/IDRs, and is intended to provide an invaluable resource for the research community for a better understanding structural disorder and for developing better computational tools for studying disordered proteins.

Intrinsic protein disorder in histone lysine methylation.

UNLABELLED: Histone lysine methyltransferases (HKMTs), catalyze mono-, di- and trimethylation of lysine residues, resulting in a regulatory pattern that controls gene expression. Their involvement in many different cellular processes and diseases makes HKMTs an intensively studied protein group, but scientific interest so far has been concentrated mostly on their catalytic domains. In this work we set out to analyze the structural heterogeneity of human HKMTs and found that many contain long intrinsically disordered regions (IDRs) that are conserved through vertebrate species. Our predictions show that these IDRs contain several linear motifs and conserved putative binding sites that harbor cancer-related SNPs. Although there are only limited data available in the literature, some of the predicted binding regions overlap with interacting segments identified experimentally. The importance of a disordered binding site is illustrated through the example of the ternary complex between MLL1, menin and LEDGF/p75. Our suggestion is that intrinsic protein disorder plays an as yet unrecognized role in epigenetic regulation, which needs to be further elucidated through structural and functional studies aimed specifically at the disordered regions of HKMTs. REVIEWERS: This article was reviewed by Arne Elofsson and Piotr Zielenkiewicz.

Efficient copper-catalyzed trifluoromethylation of aromatic and heteroaromatic iodides: the beneficial anchoring effect of borates.

Efficient copper-catalyzed trifluoromethylation of aromatic iodides was achieved with TMSCF3 in the presence of trimethylborate. The Lewis acid was used to anchor the in situ generated trifluoromethyl anion and suppress its rapid decomposition. Broad applicability of the new trifluoromethylating reaction was demonstrated in the functionalization of different aromatic and heteroaromatic iodides.

Multiple site directed mutagenesis strategy based on total RNA and RT-PCR method.

Site-directed PCR-based mutagenesis methods are widely used to generate mutations. All published methods work on DNA clones carrying the target sequence. However, DNA clones are not always available. We have previously published a RT-PCR-based site-directed mutagenesis method starting from total RNA to overcome this problem. In this article, we report an improvement of our previous method to facilitate introduction of multiple mutations into a target sequence. We demonstrate the efficacy and feasibility of this strategy by mutation of the human beta-actin gene. BamHI restriction endonuclease cleavage sites were generated within the gene to assist screening. Using three mutagenic primers in a single RT-PCR reaction, seven different clones were produced carrying three single and four multiple mutations. An investigation of the effect of the cycle number and elongation time of the PCR reactions revealed that both have an influence on the ratio of clones carrying single and multiple mutations. An optimized protocol was established for efficient multiple site-directed mutagenesis.

Multiplex site-directed mutagenesis strategy including high-efficiency selection of the mutant PCR products.

Site-directed mutagenesis is of great importance for probing the structure/function relationship of proteins. Developing our previous method (Nagy et al. Anal Biochem 324:301-303, 2004), here we report a multiplex strategy for site-directed mutagenesis using PCR in one tube to introduce a single mutation into three or more genes at the same time. DNA fragments carrying the desired mutation can be distinguished from each other in a standard antibiotic selection step of the transformed bacteria. Due to this strategy the mutagenesis procedure for several genes can be accelerated.