Mapping in silico genetic networks of the KMT2D tumour suppressor gene to uncover novel functional associations and cancer cell vulnerabilities.

Background: Loss-of-function (LOF) alterations in tumour suppressor genes cannot be directly targeted. Approaches characterising gene function and vulnerabilities conferred by such mutations are required.

Methods: Here, we computationally map genetic networks of KMT2D, a tumour suppressor gene frequently mutated in several cancer types.

Homologous recombination deficiency signatures in gastrointestinal and thoracic cancers correlate with platinum therapy duration.

There is emerging evidence about the predictive role of homologous recombination deficiency (HRD), but this is less defined in gastrointestinal (GI) and thoracic malignancies. We reviewed whole genome (WGS) and transcriptomic (RNA-Seq) data from advanced GI and thoracic cancers in the Personalized OncoGenomics trial (NCT02155621) to evaluate HRD scores and single base substitution (SBS)3, which is associated with BRCA1/2 mutations and potentially predictive of defective HRD. HRD scores were calculated by sum of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions scores.

Whole-genome and transcriptome analysis enhances precision cancer treatment options.

Background: Recent advances are enabling delivery of precision genomic medicine to cancer clinics. While the majority of approaches profile panels of selected genes or hotspot regions, comprehensive data provided by whole-genome and transcriptome sequencing and analysis (WGTA) present an opportunity to align a much larger proportion of patients to therapies.

Patients And Methods: Samples from 570 patients with advanced or metastatic cancer of diverse types enrolled in the Personalized OncoGenomics (POG) program underwent WGTA.

E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2.

The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.

Multivalent Interactions with Fbw7 and Pin1 Facilitate Recognition of c-Jun by the SCF Ubiquitin Ligase.

Many regulatory proteins, including the transcription factor c-Jun, are highly enriched in disordered protein regions that govern growth, division, survival, differentiation, and response to signals. The stability of c-Jun is controlled by poorly understood regulatory interactions of its disordered region with both the E3 ubiquitin ligase SCF and prolyl cis-trans isomerase Pin1. We use nuclear magnetic resonance and fluorescence studies of c-Jun to demonstrate that multisite c-Jun phosphorylation is required for high-affinity interaction with Fbw7.

Complex regulatory mechanisms mediated by the interplay of multiple post-translational modifications.

Post-translational modifications (PTMs), which are found largely in intrinsically disordered protein regions (IDRs), regulate protein activity, stability and interactions with partners. They are therefore critical for controlling essentially all cellular processes. A single modification event can have dramatic effects; however, proteins are often modified on multiple sites to collectively modulate the biological outcome.

An allosteric conduit facilitates dynamic multisite substrate recognition by the SCF ubiquitin ligase.

The ubiquitin ligase SCF mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2.

Dynamic Protein Interaction Networks and New Structural Paradigms in Signaling.

Understanding signaling and other complex biological processes requires elucidating the critical roles of intrinsically disordered proteins (IDPs) and regions (IDRs), which represent ∼30% of the proteome and enable unique regulatory mechanisms. In this review, we describe the structural heterogeneity of disordered proteins that underpins these mechanisms and the latest progress in obtaining structural descriptions of conformational ensembles of disordered proteins that are needed for linking structure and dynamics to function. We describe the diverse interactions of IDPs that can have unusual characteristics such as "ultrasensitivity" and "regulated folding and unfolding".

The effect of intrachain electrostatic repulsion on conformational disorder and dynamics of the Sic1 protein.

The yeast cyclin-dependent kinase inhibitor Sic1 is a disordered protein that, upon multisite phosphorylation, forms a dynamic complex with the Cdc4 subunit of an SCF ubiquitin ligase. To understand the multisite phosphorylation dependence of the Sic1:Cdc4 interaction, which ultimately leads to a sharp cell cycle transition, the conformational properties of the disordered Sic1 N-terminal targeting region were studied using single-molecule fluorescence spectroscopy. Multiple conformational populations with different sensitivities to charge screening were identified by performing experiments in nondenaturing salts and ionic denaturants.

Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding.

The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein.

Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase.

The ubiquitin ligase SCF(Cdc4) (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition.

GAP43 shows partial co-localisation but no strong physical interaction with prolyl oligopeptidase.

It has recently been proposed that prolyl oligopeptidase (POP), the cytosolic serine peptidase with neurological implications, binds GAP43 (Growth-Associated Protein 43) and is implicated in neuronal growth cone formation, axon guidance and synaptic plasticity. We investigated the interaction between GAP43 and POP with various biophysical and biochemical methods in vitro and studied the co-localisation of the two proteins in differentiated HeLa cells. GAP43 and POP showed partial co-localisation in the cell body as well as in the potential growth cone structures.

High levels of structural disorder in scaffold proteins as exemplified by a novel neuronal protein, CASK-interactive protein1.

CASK-interactive protein1 is a newly recognized post-synaptic density protein in mammalian neurons. Although its N-terminal region contains several well-known functional domains, its entire C-terminal proline-rich region of 800 amino acids lacks detectable sequence homology to any previously characterized protein. We used multiple techniques for the structural characterization of this region and its three fragments.

H-start for exclusively heteronuclear NMR spectroscopy: the case of intrinsically disordered proteins.

Here, we present a series of exclusively heteronuclear multidimensional NMR experiments, based on 13C direct detection, which exploit the (1)H polarization as a starting source to increase the signal-to-noise ratio. This contributes to make this spectroscopy more useful and usable. Examples are reported for a suitable system such as securin, an intrinsically disordered protein of 22 kDa.

Structural and dynamic characterization of intrinsically disordered human securin by NMR spectroscopy.

Understanding the molecular action of securin, the inhibitor of separase in mitosis, is of immense theoretical and biomedical importance. The residue-level structural description of an intrinsically disordered protein of this length (202 amino acids, containing 24 prolines), however, represents a particular challenge. Here we combined (1)H-detected and (13)C-detected protonless NMR experiments to achieve full assignment of securin's backbone amide resonances.

Towards proteomic approaches for the identification of structural disorder.

Intrinsically unstructured/disordered proteins (IUPs) and protein domains lack a well-defined three-dimensional structure under physiological conditions. Structural disorder imparts advantages in many non-conventional functions, which poses a significant challenge to our understanding of the structure-function relationship of proteins. The general appreciation of this fact, however, is hampered by the large gap in our knowledge on IUPs, as we have biophysical data on less than 500 of them, whereas bioinformatic predictions suggest at least several thousand such proteins in the human proteome alone.

A novel two-dimensional electrophoresis technique for the identification of intrinsically unstructured proteins.

Intrinsically unstructured proteins (IUPs) lack a well defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes, but only a handful of them have so far been identified. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization.

IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content.

Intrinsically unstructured/disordered proteins and domains (IUPs) lack a well-defined three-dimensional structure under native conditions. The IUPred server presents a novel algorithm for predicting such regions from amino acid sequences by estimating their total pairwise interresidue interaction energy, based on the assumption that IUP sequences do not fold due to their inability to form sufficient stabilizing interresidue interactions. Optional to the prediction are built-in parameter sets optimized for predicting short or long disordered regions and structured domains.