ELM-the eukaryotic linear motif resource in 2020.

The eukaryotic linear motif (ELM) resource is a repository of manually curated experimentally validated short linear motifs (SLiMs). Since the initial release almost 20 years ago, ELM has become an indispensable resource for the molecular biology community for investigating functional regions in many proteins. In this update, we have added 21 novel motif classes, made major revisions to 12 motif classes and added >400 new instances mostly focused on DNA damage, the cytoskeleton, SH2-binding phosphotyrosine motifs and motif mimicry by pathogenic bacterial effector proteins. The current release of the ELM database contains 289 motif classes and 3523 individual protein motif instances manually curated from 3467 scientific publications. ELM is available at: http://elm.eu.org

Visual analysis of protein-ligand interactions

The analysis of protein-ligand interactions is complex because of the many factors at play. Most current methods for visual analysis provide this information in the form of simple 2D plots, which, besides being quite space hungry, often encode a low number of different properties. In this paper we present a system for compact 2D visualization of molecular simulations. It purposely omits most spatial information and presents physical information associated to single molecular components and their pairwise interactions through a set of 2D InfoVis tools with coordinated views, suitable interaction, and focus+context techniques to analyze large amounts of data. The system provides a wide range of motifs for elements such as protein secondary structures or hydrogen bond networks, and a set of tools for their interactive inspection, both for a single simulation and for comparing two different simulations. As a result, the analysis of protein-ligand interactions of Molecular Simulation trajectories is greatly facilitated.Peer ReviewedPostprint (author's final draft

A structural biology community assessment of AlphaFold2 applications

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods for protein structure predictions have reached the accuracy of experimentally determined models. Although this has been independently verified, the implementation of these methods across structural-biology applications remains to be tested. Here, we evaluate the use of AlphaFold2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modeling of interactions; and modeling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modeled when compared with homology modeling, identifying structural features rarely seen in the Protein Data Bank. AF2-based predictions of protein disorder and complexes surpass dedicated tools, and AF2 models can be used across diverse applications equally well compared with experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life-science research

New Insights into the Mechanisms Underlying NEDD8 Structural and Functional Specificities

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins are small polypeptides that are conjugated to substrates affecting their activity and stability. Cells encode “receptors” containing Ub-/Ubl-binding domains that interpret and translate each modification into appropriate cellular responses. Among the different Ubls, NEDD8, which is the ubiquitin’s closest relative, retains many of the structural determinants that enable ubiquitin the ability to target proteins to degradation. Nevertheless, the direct involvement of NEDD8 conjugation to proteasome recruitment has been proved only in a few cases. To date, well-defined major NEDD8 substrates are primarily members of the cullin family, and cullin neddylation does not appear to mark these proteins for degradation. Various studies have demonstrated that selectivity between ubiquitin and NEDD8 is guaranteed by small but substantial differences. Nevertheless, several issues still need to be addressed, mainly concerning which interaction surfaces mediate NEDD8 function and what domains recognize them. Recently, two novel domains identified in KHNYN and N4BP1 proteins have shed new light on this research area. Here, I discuss some recent reports that contributed to shed light on the mechanisms underlining the discrimination between ubiquitin and NEDD8. Understanding the details of these molecular mechanisms represents a prominent facet for the identification of new therapeutic targets

Hereditary Hearing Loss: From Molecular Bases To Phenotypic Caractherization

Hearing loss is one of the most common birth defects in developed countries. Approximately one/two in 1000 newborns are diagnosed with bilateral permanent sensorineural hearing loss. Hereditary hearing loss can be syndromic (about 25%), in which deafness is associated with other signs and/or symptoms, and non-syndromic (about 75%), in which no other clinical features are present. Non-syndromic hearing loss (NSHL) is characterized by a vast genetic heterogeneity with more than 160 loci described in humans and 100 genes so far identified. NSHL generally follows simple Mendelian inheritance and is predominantly transmitted as an autosomal recessive trait (75-80%), although other modes of inheritance are possible: autosomal dominant (20%), X-linked (2-5%) and mitochondrial (1%). Given the high genetic heterogeneity of HL, tests based on NGS technologies are rapidly replacing many single-gene Sanger sequencing tests, due to their technical limits and higher costs. The aim of this work was translational, with the goal to develop advanced molecular tools, with high diagnostic rate, and to investigate the genetic bases of NSHL in a population of Caucasian individuals, mainly of pediatric age. A customized NGS targeted panel of 59 genes, strongly associated, in Caucasians, with NSHL or with SHL, which onset is usually characterized by isolated deafness (i.e. Pendred and Usher syndrome), was designed and developed. The Ion Torrent PGMTM platform and a customized bioinformatics pipeline have been used for the analysis of DNA samples collected from clinically highly selected subjects with a previous negative test for the frequently mutated GJB2 gene. A series of 78 cases has undergone a complete study; an etiological diagnosis has been established for 34 of these subjects, with an overall diagnostic yield of 43.6%. For each tested subject, an average depth of coverage of 249 X in the analyzed sequences and a mean of 499 variants were obtained. Likely causative identified variants were located in the following 20 genes: CDH23, GJB2, COCH, MYO7A, ADGRV1, EYA4, OTOG, SLC17A8, TMPRSS3, ACTG1, CEACAM16, COL11A2, GJB3, KCNQ4, MYH9, MYO6, PTPRQ, SLC26A4, STRC, TMC1. The most frequently mutated gene in our cohort was CDH23, which, even in our cases, accounted both for NSHL and Usher syndrome type 1 phenotypes. A novel EYA4 mutation, identified in two related subjects with post-lingual progressive deafness, was found to co-segregate in two individuals of the same family, with a Waardenburg syndrome phenotype, due to a novel PAX3 gene mutation. All the identified variants were collected in an in-house database that proved an invaluable tool for the identification of recurrent variants or possible alignment errors, and for further stratification and correlation between genotype and phenotype. In conclusion, the targeted gene-panel we have developed, in combination with the in-house bioinformatics pipeline, is a proven sensitive diagnostic tool capable of providing an extremely competitive diagnostic yield. Our work further demonstrates the importance of integrating the power of NGS technology and data process with the fundamental role of a strong clinical evaluation in keeping with what is expected from modern medicine

Investigating how ubiquitin-mediated proteolysis of AURKA contributes to its activity in the cell cycle

Aurora kinase A (AURKA) is a major mitotic regulatory kinase required for mitotic entry, the formation of a bipolar mitotic spindle, and the completion of cytokinesis. In recent years AURKA has been identified as an upstream regulator for many interphase functions such as cilia disassembly and mitochondrial fragmentation. AURKA is overexpressed in many tumours and has a pivotal role in the acquisition of malignant cell phenotypes. Therefore, it is considered a highly attractive drug target for anti-cancer therapy. The activity of AURKA is regulated by phosphorylation at the active loop or the interaction with binding partners. TPX2 is a well-known binding partner of AURKA. It activates AURKA through stabilizing the T-loop and is required for targeting AURKA to the mitotic spindle. Phosphorylation and binding partners may act synergistically to induce hyperactivity of the kinase. Previous research from my lab has highlighted that AURKA is frequently co-expressed with TPX2 in human cancers and proposed AURKA/TPX2 complex as an oncogenic holoenzyme in a variety of cancers. AURKA protein is targeted for proteasome-mediated degradation by the FZR1 activated form of APC/C at the end of mitosis. This study focuses on characterisation of AURKA degrons, the contribution of APC/C-FZR1 in the timing of AURKA inactivation, identifying the physiological consequences of AURKA deregulation outside mitosis, and examining the role of Short Linear motifs (SLiMs) within AURKA N-terminal domain in regulating its stability and activity. I show that the previously known D-box-like motif (R371xxL374) within C-terminal is not a functional degron. I also reveal that the A-box motif may act as an atypical D-box that is sufficient to drive protein degradation. I use a new tool CRISPR/Cas9 FZR1 knockout cell line and a FRET-based biosensor for measuring AURKA activity to investigate directly whether AURKA inactivation is regulated simply by destruction. These, in combination with time-lapse imaging, show that inactivation of AURKA is identical in wild-type and FZR1KO cells, despit¬e the difference in protein levels between the two cell lines. I demonstrate that the timing of AURKA inactivation is regulated via the degradation of its activator TPX2 at mitotic exit. Moreover, the destruction of AURKA is required to regulate its interphase activity. I also identify that extra AURKA activity can have consequences on the morphology of the mitochondrial network outside of mitosis. My time-lapse imaging reveals that FZR1-restricted degradation of AURKA controls mitochondrial dynamics. This mechanism links the destruction machinery, through AURKA signaling to the mitochondrial dynamics of the cell. I further explore the role of the N-terminal domain in the regulation of AURKA activity through the detailed analysis of the potential SLiMs. I find that K23RVL has a role in mediating the autoinhibition of AURKA. I then investigate the hypothesis that calmodulin (CaM) protects AURKA from degradation through its binding to the A-box SLiM. I find that AURKA degradation is not affected by inhibition of Ca2+/CaM. In summary, this work sheds light not only on the molecular mechanisms of AURKA activity and stability but also on the physiological relevance outside mitosis, which is urgently needed in the field to understand the oncogenic activity of AURKA and to improve therapeutic applications of cancer patients.Cambridge Overseas Trust, Youssef Jameel Foundation and Cambridge Philosophical Society

Examining the mechanistic regulation of starvation-induced autophagy via the identification and characterisation of novel ULK kinase substrates

Autophagy involves the formation of an endoplasmic reticulum-derived membrane termed a phagophore which expands to engulf cytoplasmic cargo before sealing to form an autophagosome. Amino acid starvation is amongst the most potent autophagic stimuli, however whilst the key signalling complexes involved in starvation-induced autophagy are known, the precise regulatory mechanisms remain poorly understood. The serine/threonine kinase ULK1 and close homolog ULK2 assume the most upstream position in the autophagic signalling cascade and play a crucial yet enigmatic role in coordinating the autophagic machinery. To further understand the mechanisms of starvation-induced autophagy, I performed a number of unbiased phosphoproteomic screens to identify ULK substrates before classifying their roles in starvation-induced autophagy. Analysis of these datasets has revealed that loss of ULK results in significant changes to the phosphoproteome and has yielded a high confidence list of potential substrates whilst also offering interesting insights into the veracity of the published ULK consensus signature. Amongst the novel phosphorylation targets are components of the retromer and AMPK complexes along with multiple components of the class III PI3K VPS34 complex. The pseudokinase p150, scaffolding component of the VPS34 complex, is phosphorylated by ULK1 in vitro and in vivo at serine 861. CRISPR-based knockout of p150 results in inhibition of autophagy and endosomal trafficking, whilst mutating the phosphorylated residue in p150 alters both omegasome establishment and autophagic flux. Furthermore, incorporation of phosphomutant p150 into the VPS34 complex modulates its lipid kinase activity in vitro. These data identify a novel ULK-dependent signalling axis and help illuminate the complexities of signal transduction in autophagy