Proteomic analyses reveal misregulation of LIN28 expression and delayed timing of glial differentiation in human iPS cells with MECP2 loss-of-function.

Rett syndrome (RTT) is a pervasive developmental disorder caused by mutations in MECP2. Complete loss of MECP2 function in males causes congenital encephalopathy, neurodevelopmental arrest, and early lethality. Induced pluripotent stem cell (iPSC) lines from male patients harboring mutations in MECP2, along with control lines from their unaffected fathers, give us an opportunity to identify some of the earliest cellular and molecular changes associated with MECP2 loss-of-function (LOF). We differentiated iPSC-derived neural progenitor cells (NPCs) using retinoic acid (RA) and found that astrocyte differentiation is perturbed in iPSC lines derived from two different patients. Using highly stringent quantitative proteomic analyses, we found that LIN28, a gene important for cell fate regulation and developmental timing, is upregulated in mutant NPCs compared to WT controls. Overexpression of LIN28 protein in control NPCs suppressed astrocyte differentiation and reduced neuronal synapse density, whereas downregulation of LIN28 expression in mutant NPCs partially rescued this synaptic deficiency. These results indicate that the pathophysiology of RTT may be caused in part by misregulation of developmental timing in neural progenitors, and the subsequent consequences of this disruption on neuronal and glial differentiation

Protein-protein interaction confidence assessment and network clustering computational analysis

Protein-protein interactions represent a crucial source of information for the understanding of the biological mechanisms of the cell. In order to be useful, high quality protein-protein interactions must be computationally extracted from the noisy datasets produced by high-throughput experiments such as affinity purification. Even when filtered protein-protein interaction datasets are obtained, the task of analyzing the network formed by these numerous interactions remains tremendous. Protein-protein interaction networks are large, intricate, and require computational approaches to provide meaningful biological insights. The overall objective of this thesis is to explore algorithms assessing the quality of protein-protein interactions and facilitating the analysis of their networks. This work is divided into four results: 1) a novel Bayesian approach to model contaminants originating from affinity purifications, 2) a new method to identify and evaluate the quality of protein-protein interactions independently in different cell compartments, 3) an algorithm computing the statistical significance of clusterings of proteins sharing the same functional annotation in protein-protein interaction networks, and 4) a computational tool performing sequence motif discovery in 5' untranslated regions as well as evaluating the clustering of such motifs in protein-protein interaction networks.Les interactions protéine-protéine représentent une source d'information essentielle à la compréhension des divers méchanismes biologiques de la cellule. Cependant, les expériences à haut débit qui identifient ces interactions, comme la purification par affinité, produisent un très grand nombre de faux-positifs. Des méthodes computationelles sont donc requises afin d'extraire de ces ensembles de données les interactions protéine-protéine de grande qualité. Toutefois, même lorsque filtrés, ces ensembles de données forment des réseaux très complexes à analyser. Ces réseaux d'interactions protéine-protéine sont d'une taille importante, d'une grande complexité et requièrent des approches computationelles sophistiquées afin d'en retirer des informations possédant une réelle portée biologique. L'objectif de cette thèse est d'explorer des algorithmes évaluant la qualité d'interactions protéine-protéine et de faciliter l'analyse des réseaux qu'elles composent. Ce travail de recherche est divisé en quatre principaux résultats: 1) une nouvelle approche bayésienne permettant la modélisation des contaminants provenant de la purification par affinité, 2) une nouvelle méthode servant à la découverte et l'évaluation de la qualité d'interactions protéine-protéine à l'intérieur de différents compartiments de la cellule, 3) un algorithme détectant les regroupements statistiquement significatifs de protéines partageant une même annotation fonctionnelle dans un réseau d'interactions protéine-protéine et 4) un outil computationel qui a pour but la découverte de motifs de séquences dans les régions 5' non traduites tout en évaluant le regroupement de ces motifs dans les réseaux d'interactions protéine-protéine

A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity.

Methylation is a post-translational modification that can affect numerous features of proteins, notably cellular localization, turnover, activity, and molecular interactions. Recent genome-wide analyses have considerably extended the list of human genes encoding putative methyltransferases. Studies on protein methyltransferases have revealed that the regulatory function of methylation is not limited to epigenetics, with many non-histone substrates now being discovered. We present here our findings on a novel family of distantly related putative methyltransferases. Affinity purification coupled to mass spectrometry shows a marked preference for these proteins to associate with various chaperones. Based on the spectral data, we were able to identify methylation sites in substrates, notably trimethylation of K135 of KIN/Kin17, K561 of HSPA8/Hsc70 as well as corresponding lysine residues in other Hsp70 isoforms, and K315 of VCP/p97. All modification sites were subsequently confirmed in vitro. In the case of VCP, methylation by METTL21D was stimulated by the addition of the UBX cofactor ASPSCR1, which we show directly interacts with the methyltransferase. This stimulatory effect was lost when we used VCP mutants (R155H, R159G, and R191Q) known to cause Inclusion Body Myopathy with Paget's disease of bone and Fronto-temporal Dementia (IBMPFD) and/or familial Amyotrophic Lateral Sclerosis (ALS). Lysine 315 falls in proximity to the Walker B motif of VCP's first ATPase/D1 domain. Our results indicate that methylation of this site negatively impacts its ATPase activity. Overall, this report uncovers a new role for protein methylation as a regulatory pathway for molecular chaperones and defines a novel regulatory mechanism for the chaperone VCP, whose deregulation is causative of degenerative neuromuscular diseases

KIN, VCP, and a number of hsp70 isoforms are each trimethylated on lysine residues by specific methyltransferases within this family.

<p>(A) Linear representation of all identified substrates with domain architecture. Residues delineating each domain are marked below. ZnF, Zinc Finger; WH, Winged Helix; SH3, Src Homology 3; SBD, substrate binding domain. Position of the methylated lysines is shown above. (B) Multiple sequence alignment of the region surrounding trimethylated lysines (boxed) in human VCP, KIN, and Hsp70 isoforms compared to paralogous genes in various organisms. Hs, <i>Homo sapiens</i>; Sc, <i>Saccharomyces cerevisiae</i>; Dm, <i>Drosophila melanogaster</i>; At, <i>Arabidopsis thaliana</i>; Pf, <i>Plasmodium falciparum</i>. Color code is as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003210#pgen-1003210-g001" target="_blank">Figure 1</a>. Strong and weak residue similarity are represented by a colon (:) and period (.), respectively, and asterisk (*) denotes identity. (C–E) In vitro methylation assays with tritium-labeled S-adenosylmethionine of KIN-His with GST-METTL22 (C), VCP-His with GST-METTL21D (D), and three His-tagged hsp70 isoforms (HSPA1, HSPA5, and HSPA8) with GST-METTL21A (E). In each case, substitution of the methylated lysine by an arginine leads to loss of methylation signal as detected by autoradiography. Coomassie staining of the gel shows total proteins loaded onto the gel and serves as control.</p

ASPSCR1 promotes methylation of VCP by METTL21D.

<p>(A) In vitro methylation assays of VCP-His, UBXN6-His, ASPSCR1-His and fragments of ASPSCR1 with GST-METTL21D. Various combinations of the UBX proteins were added to reactions containing VCP. Coomassie staining of the gel is shown. (B) Linear representation of ASPSCR1 showing domain architecture of the protein (UBL, UBiquitin-Like domain; SHP, SHP box; UBX, UBiquitin regulatory X domain; CC, Coiled-Coil domain) and localization of residue 280 which marks the boundary between the N- and C- terminal fragments used in these experiments. (C) In vitro GST pull-down assays of VCP-His, UBXN6-His, ASPSCR1-His and fragments of ASPSCR1 with GST-METTL21D. Combinations of full-length ASPSCR1 and its fragments were once again employed with VCP. (D) Linear representation of VCP showing domain architecture of the protein (including double Ψ barrel superfold and 4-stranded β barrel of the N-terminal domain, Walker A and B motifs, as well as 4 α helices bundle of ATPase domains D1 and D2 as well as linker regions L1 and L2 and C-terminal domain) and localization of the mutants used in this study as well as the site of methylation. (E) In vitro methylation assays of wild-type VCP-His and IBMPFD and ALS-causing mutations R155H, R159G and R191Q in presence or absence of ASPSCR1-His. (F) In vitro GST pull-down assays of the same combination of proteins as in (E).</p

Tandem-affinity purification coupled to mass spectrometry reveals protein interaction network of putative methyltransferases.

<p>Purification of 10 TAP-tagged putative methyltransferases from ponasterone-inducible strains of HEK 293 cells. Eluted proteins were separated by SDS-PAGE. Gels were silver stained and cut in slices that were then trypsin digested before protein identification by LC-MS/MS. Tagged baits and major interactors are marked.</p

Computational analysis defines a novel family of putative protein methyltransferases.

<p>(A) Unrooted phylogenetic tree of a family of human putative methyltransferases distantly related to PRMTs. FAM86 represents a number of genetic variants (FAM86A, B1, B2, C, and D) whose duplication is observed only in primates. Branch lengths are not proportional to the actual evolutionary distances between the sequences. (B) Secondary structure organization of the Rossmann fold domain of PRMTs responsible for the methyltransferase activity. Arrows represent β strands and rectangles correspond to α helices, including typically ill-defined or inexistent α helix C. (C) Multiple sequence alignment of the Rossmann fold of all members within this family as generated by ClustalW2 (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). Red residues are small, hydrophobic, aromatic; blue are acidic; magenta are basic; and all other residues are green. Primary sequence alignment corresponds nicely with secondary structure prediction by Jpred3 (<a href="http://www.compbio.dundee.ac.uk/www-jpred/" target="_blank">http://www.compbio.dundee.ac.uk/www-jpred/</a>). Overhead β strands and α helices are shown as in (B). Conserved motif I, the site of S-adenosylmethionine binding, is also marked.</p

Methylation of VCP decreases the activity of its N-terminal ATPase domain.

<p>(A) Linear representation of a fragment of VCP encompassing its N-terminal and first ATPase domain employed in the ATPase assay. Proximity of the methylated lysine to the Walker B motif is highlighted above. (B) In vitro methylation assays of 1–481_VCP-His fragment by GST-METTL21D as compared to full length VCP. (C, D) Colorimetric assays to measure released phosphate (C) and relative ATPase activity (D) by the 1–481 fragment of VCP. The experiment was done in triplicate. Data from the last 3 time points (9 measurements in total for each condition) was compiled to generate the graph shown in (D). (E) In vitro GST pull-down assay of VCP-His with GST-METTL21D. In all experiments the effect of un methylatable VCP mutant K135R, catalytically inactive METTL21D mutant E73Q, and methylation inhibitor S-adenosylhomocysteine is shown.</p

Polyphosphate kinase regulates LPS structure and polymyxin resistance during starvation in E. coli.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK