Variable glutamine-rich repeats modulate transcription factor activity

Excessive expansions of glutamine (Q)-rich repeats in various human proteins are known to result in severe neurodegenerative disorders such as Huntington's disease and several ataxias. However, the physiological role of these repeats and the consequences of more moderate repeat variation remain unknown. Here, we demonstrate that Q-rich domains are highly enriched in eukaryotic transcription factors where they act as functional modulators. Incremental changes in the number of repeats in the yeast transcriptional regulator Ssn6 (Cyc8) result in systematic, repeat-length-dependent variation in expression of target genes that result in direct phenotypic changes. The function of Ssn6 increases with its repeat number until a certain threshold where further expansion leads to aggregation. Quantitative proteomic analysis reveals that the Ssn6 repeats affect its solubility and interactions with Tup1 and other regulators. Thus, Q-rich repeats are dynamic functional domains that modulate a regulator's innate function, with the inherent risk of pathogenic repeat expansions

Deglycating enzymes in plant and prokaryotes

On sait depuis longtemps que les amines, y compris celles des protéines, réagissent spontanément avec les sucres réducteurs. La base de Schiff qui se forme dans un premier temps se réarrange spontanément en une cétosamine ou produit d’Amadori. Dans les cellules mammaliennes, l’exemple le plus connu de cette réaction, dite de glycation, est celle du glucose avec les protéines, entraînant la formation de fructosamines. Par ailleurs, cette formation est incriminée dans le développement de complications à long terme du diabète entre autres parce que les fructosamines sont spontanément converties en molécules toxiques dites produits avancés de glycation. Nous savons depuis quelques années que les fructosamines sont métabolisées dans les cellules. La fructosamine-3-kinase (FN3K) est une enzyme mammalienne dont le rôle est de phosphoryler les fructosamines. Les fructosamines-3-phosphates ainsi formées sont instables et se décomposent spontanément avec régénération de l’amine initiale. De ce fait, la FN2K est responsable d’un mécanisme de réparation des protéines. Dans le génome humain, il existe une protéine présentant 65% d’identité avec le FN3K et dénommée ‘fructosamine-3-kinase-related protein’ (FN3K-RP). Cette enzyme ne phosphoryle pas les fructosamines, mais d’autres produits d’Amadori, principalement les ribulosamines et les érythrulosamines. La présence des fructosamines est déjà démontrée in vivo ; par contre celle des ribulosamines et érythrulosamines n’a pas encore été démontrée. Les ribulosamines et les érythrulosamines ne se forment vraisemblablement pas à partir de ribose et d’érythrose libre, mais à partir de ribose-5-phosphate et d’érythrose-4-phosphate, deux monosaccharides présents dans toutes les cellules et qui possèdent un pouvoir glycant élevé. Les ribulosamines-5-phosphates et les érythrusolamines-4-phosphates ainsi formées ne sont pas des substrats pour la FN3K-RP et doivent être préalablement déphosphorylées. Une phosphatase agissant sur les ribulosamines-5-phosphates a été purifiée à partir de globules rouges humains et identifiée comme étant la ‘low-molecular-weigtht protein-tyrosine-phosphatase-A’ (LMW-PTP-A). Le but de ce travail est d’examiner la fonction des homologues de la FN3K de plantes et des bactéries. Une attention particulière a été accordée aux espèces bactériennes dans le but de trouver des protéines fonctionnellement liées à l’homologue de la FN3K en recherchant des opérons contenant celui-ci. Dans l’introduction, nous passons en revue les mécanismes de formation des produits d’Amadori. Ceux-ci peuvent se former spontanément avec toutes sorts d’amines et de sucres comme mentionnés plus haut. Ils peuvent être également synthétisés enzymatiquement à partir de glucose-6-phosphate, pour former certaines opines, ou à partir de dérivés du ribose-5-phosphate, dans le cadre de la synthèse de l’histidine, du trytophane et de ptéridines. Nous passons aussi en revue les enzymes impliquées dans la dégradation du produits d’Amadori, à savoir (1) les fructosyl-aminoacides oxydases, (2) les enzymes de la voie des fructosamines-6-phosphates et (3) la fructosamine-3-kinase et les enzymes qui lui sont fonctionnellement apparentées. Les résultats comportent 5 parties : En premier lieu, l’homologue de la FN3K d’Arabidopsis thaliana a été exprimé, purifié et caractérisé (Chapitre 1 des résultats). Cette protéine phosphoryle les ribulosamines et les érythrulosamines, mais pas les fructosamines. Elle possède donc une activité identique à l’activité purifiée à partir de feuilles d’épinard. Ceci indique que l’homologue de la FN3K de plantes, qui est vraisemblablement présent dans les chloroplastes, possède une activité ribulosamine/érythrulosamine-3-kinase comparable à celle de la FN3K-RP humaine. Des homologues de la FN3K ont été aussi identifiés dans ≈ 230 (soit ≈ 14%) des 1652 génomes bactériens séquencés. Dans 16 génomes, appartenant à des espèces phylogénétiquement distantes, l’homologue de la FN3K forme un opéron avec un homologue de la LMW-PTP, ce qui suggère que ces deux gènes sont fonctionnellement liés (Chapitre 2). Nous avons par la suite exprimé, purifié et caractérisé six homologues de la FN3K appartenant aux génomes d’Escherichia coli, Enterococcus faecium, Lactobacillus plantarum, Salmonella typhimurium, Staphylococcus aureus et Thermus thermophilus (Chapitre 3). A l’exception des homologues d’E. coli et de S. typhimurium, les homologues bactériens de la FN3K ont une activité ribulosamine/érythrulosamine-3-kinase. Ces enzymes sont actives sur les dérivés de la lysine, mais pas sur les fructosamines, ni sur les ribulosamines liées à l’amine alpha de différents acides aminées. Ils phosphorylent également les ribulosamines et les érythrulosamines liées aux protéines. Leurs propriétés ressemblent donc à celles de la FN3K-RP humaine. Les homologues de la FN3K d’E. coli et de S. typhimurium sont inactifs sur tous les substrats que nous avons testés et ont probablement acquis une spécificité de substrat différente de celle de la famille des protéines de la FN3K. Nous avons également exprimé, purifié et caractérisé l’homologue de la LMW-PTP qui forme un opéron avec l’homologue de la FN3K dans le génome de T. thermophilus, ainsi que les deux homologues de la LMW-PTP de S. aureus (PtpA et PtpB), dont aucun des deux ne forme d’opéron avec l’homologue de la FN3K (Chapitre 4). L’homologue de la LMW-PTP de T. thermophilus ainsi qu’un homologue de S. aureus (PtpA) agissent non seulement comme des protéines-tyrosine-phosphostases, mais aussi comme des protéines-ribulosamine-5-phosphatases et érythruloselysine-4-phosphate. De plus, en déphosphorylant les ribulosamines-5-phosphates et les érythrulosamines-4-phosphates, ces homologues de la LMW-PTP forment des substrats pour les homologues bactériens de la FN3K. Les enzymes bactériennes ont été utilisées pour la détection des ribulosamines dans des tissus de mammifères (Chapitre 5). Des ribulosamines ont été détectées sur la ‘myelin basic protein’ bovine commerciale, mais leur présence était vraisemblablement les résultats d’une modification post-mortem. Ces résultats suggèrent que les homologues bactériens de la FN3K peuvent être utilisés pour détecter des ribulosamines. En conclusion, nous avons montré que l’homologue de la FN3K de plante et la plupart des homologues bactériens de la FN3K sont des ribulosamines/érythrulosamines-3-kinase. Ces enzymes pourraient agir avec une phosphatase pour initier la réparation de produits de glycation formés à a partir de ribose-5-phosphate et d’érythrose-4-phosphate. Dans la discussion, nous analysons également les propriétés de ka FN3K et de ses homologues en prenant en compte la structure cristallographique de l’homologue de la FN3K de Thermobifida fusca, récemment déposée dans la banque PDBGlycation is the term given to describe the spontaneous reaction of reducing sugars with amines. This reaction yields a ketoamine, or Amadori product. In mammalian cells the best-known example of a glycation reaction takes place between glucose, the prevalent monosaccharide, and amino groups of proteins, giving rise to fructosamines. These are precursors for more complex molecules called advanced glycation end-products, which are thought to be implicated in complications of diabetes and in the ageing process. Contrary to what was long believed, fructosamines can be metabolised inside the cells. Fructosamine 3-kinase (FN3K) is a mammalian enzyme that catalyses the phosphorylation of fructosamines. This leads to their destabilisation and removal from proteins. Thus FN3K is responsible for a new form of protein repair called deglycation. A related mammalian enzyme called fructosamine 3-kinase-related protein (FN3K-RP), sharing 65 % sequence identity with FN3K, does not phosphorylate fructosamines but other Amadori products, mainly ribulosamines and erythrulosamines. The sources of ribulosamines and erythrulosamines are probably not free ribose and erythrose but rather ribose 5-phosphate and erythrose 4-phosphate, two potent glycating agents that are present in all cell types. The resulting ribulosamine 5-phosphates and erythrulosamine 4-phosphates are first dephosphorylated by a phosphatase identified as low-molecular-weight protein-tyrosine-phosphatase-A (LMW-PTP-A), which converts these compounds to subtrates for FN3K-RP. The general goal of this thesis was to explore the function of FN3K homologues in plant and bacterial species. Particular emphasis was given to bacteria in an effort to find functionally related proteins to the FN3K homologue through the search for operons comprising a FN3K homologue. Firstly, the Arabidopsis thaliana FN3K homologue was overexpressed in Escherichia coli, purified and characterised. This protein phosphorylates ribulosamines and erythrulosamines but not fructosamines, similarly to the ribulosamine 3-kinase activity initially purified from spinach leaf extracts. This indicates that the plant FN3K homologue, which appears to be targeted to chloroplasts, is a ribulosamine/erythrulosamine 3-kinase having similar properties to mammalian FN3K-RP. Secondly, FN3K homologues were identified in ¡Ö 230 (i.e. ¡Ö 14 %) of the sequenced bacterial genomes. In 16 of these genomes, from phylogenetically distant bacteria, the FN3K homologue is immediately preceded by a LMW-PTP homologue, which is therefore probably functionally related to the FN3K homologue. Six bacterial FN3K homologues (from E. coli, Enterococcus faecium, Lactobacillus plantarum, Salmonella typhimurium, Staphylococcus aureus, and Thermus thermophilus) were overexpressed in E. coli, purified, and their kinetic properties investigated. Four of them were ribulosamine/erythrulosamine 3-kinases acting best on free lysine and cadaverine derivatives, but not on ribulosamines bound to the alpha amino group of amino acids. They also phosphorylated protein-bound ribulosamines or erythrulosamines but not protein-bound fructosamines, having therefore properties similar to those of mammalian FN3K-RP. The E. coli and S. typhimurium FN3K homologues were inactive on all tested substrates and could have therefore acquired a different substrate specificity from that of the other members of the FN3K family of proteins. The LMW-PTP homologue of T. thermophilus, which forms an operon with a FN3K homologue, and the two LMW-PTP homologues of S. aureus (PtpA and PtpB), which do not form operons with the FN3K homologue, were overexpressed in E. coli, purified and characterised. The T. thermophilus LMW-PTP homologue and one of the S. aureus LMW-PTPs (PtpA) dephosphorylated not only protein tyrosine-phosphates, but also protein ribulosamine 5-phosphates and erythrulosamine 4-phosphates, as well as free ribuloselysine 5-phosphate and erythruloselysine 4-phosphate converting them into substrates for the FN3K homologues. The plant FN3K homologue and most bacterial FN3K homologues are ribulosamine/erythrulosamine 3-kinases. They may serve, in conjunction with a phosphatase, to deglycate products of glycation formed from ribose 5-phosphate or erythrose 4-phosphate.Thèse de doctorat en sciences biomédicales et pharmaceutiques (SBIM 3) -- UCL, 200

How to prepare and deliver a great talk

Giving a talk can be daunting, particularly for scientists at the very early stages of their careers. Standing in front of an audience and speaking for 20?30 minutes do is never easy, and even those who do it for a liv- ing often confess to getting nervous before a talk. It is an oft-quoted fact that public speaking ranks higher than dying or jumping from a plane (with a parachute we hasten to add) on lists of ?what people fear the most?. Anyone who has experienced the fear of public speaking knows very well just how stomach-churning the thought of an upcoming talk can be; the dread, the sleepless nights, the thought that nothing will come out when you open your mouth, or even worse, that you will speak complete gibberish. If you have felt this way before giving a talk, do not worry, you are not alone, we have all had these fears at some stag

Variable tandem repeats accelerate evolution of coding and regulatory sequences

Genotype-to-phenotype mapping commonly focuses on two major classes of mutations: single nucleotide polymorphisms (SNPs) and copy number variation (CNV). Here, we discuss an underestimated third class of genotypic variation: changes in microsatellite and minisatellite repeats. Such tandem repeats (TRs) are ubiquitous, unstable genomic elements that have historically been designated as nonfunctional "junk DNA" and are therefore mostly ignored in comparative genomics. However, as many as 10% to 20% of eukaryotic genes and promoters contain an unstable repeat tract. Mutations in these repeats often have fascinating phenotypic consequences. For example, changes in unstable repeats located in or near human genes can lead to neurodegenerative diseases such as Huntington disease. Apart from their role in disease, variable repeats also confer useful phenotypic variability, including cell surface variability, plasticity in skeletal morphology, and tuning of the circadian rhythm. As such, TRs combine characteristics of genetic and epigenetic changes that may facilitate organismal evolvability.status: publishe

Beyond Junk: Variable tandem repeats as facilitators of rapid evolution of regulatory and coding sequences

Copy Number Variations (CNVs) and Single Nucleotide Polymorphisms (SNPs) have been the major focus of most large-scale comparative genomics studies to date. Here, we discuss a third, largely ignored, type of genetic variation, namely changes in tandem repeat number. Historically, tandem repeats have been designated as non functional "junk" DNA, mostly as a result of their highly unstable nature. With the exception of tandem repeats involved in human neurodegenerative diseases, repeat variation was often believed to be neutral with no phenotypic consequences. Recent studies, however, have shown that as many as 10% to 20% of coding and regulatory sequences in eukaryotes contain an unstable repeat tract. Contrary to initial suggestions, tandem repeat variation can have useful phenotypic consequences. Examples include rapid variation in microbial cell surface, tuning of internal molecular clocks in flies and the dynamic morphological plasticity in mammals. As such, tandem repeats can be useful functional elements that facilitate evolvability and rapid adaptation.status: publishe

Beyond Junk-Variable Tandem Repeats as Facilitators of Rapid Evolution of Regulatory and Coding Sequences

gene

Plant ribulosamine/erythrulosamine 3-kinase, a putative protein-repair enzyme.

FN3K (fructosamine 3-kinase) is a mammalian enzyme that catalyses the phosphorylation of fructosamines, which thereby becomes unstable and detaches from proteins. The homologous mammalian enzyme, FN3K-RP (FN3K-related protein), does not phosphorylate fructosamines but ribulosamines, which are probably formed through a spontaneous reaction of amines with ribose 5-phosphate, an intermediate of the pentose-phosphate pathway and the Calvin cycle. We show in the present study that spinach leaf extracts display a substantial ribulosamine kinase activity (approx. 700 times higher than the specific activity of FN3K in erythrocytes). The ribulosamine kinase was purified approx. 400 times and shown to phosphorylate ribulose-epsilon-lysine, protein-bound ribulosamines and also, with higher affinity, erythrulose-epsilon-lysine and protein-bound erythrulosamines. Evidence is presented for the fact that the third carbon of the sugar portion is phosphorylated by this enzyme and that this leads to the formation of unstable compounds decomposing with half-lives of approx. 30 min at 37 degrees C (ribulosamine 3-phosphates) and 5 min at 30 degrees C (erythrulosamine 3-phosphates). This decomposition results in the formation of a 2-oxo-3-deoxyaldose and inorganic phosphate, with regeneration of the free amino group. The Arabidopsis thaliana homologue of FN3K/FN3K-RP was overexpressed in Escherichia coli and shown to have properties similar to those of the enzyme purified from spinach leaves. These results indicate that the plant FN3K/FN3K-RP homologue, which appears to be targeted to the chloroplast in many species, is a ribulosamine/erythrulosamine 3-kinase. This enzyme may participate in a protein deglycation process removing Amadori products derived from ribose 5-phosphate and erythrose 4-phosphate, two Calvin cycle intermediates that are potent glycating agents

Many fructosamine 3-kinase homologues in bacteria are ribulosamine/erythrulosamine 3-kinases potentially involved in protein deglycation

The purpose of this work was to identify the function of bacterial homologues of fructosamine 3-kinase (FN3K), a mammalian enzyme responsible for the removal of fructosamines from proteins. FN3K homologues were identified in approximately 200 (i.e. approximately 27%) of the sequenced bacterial genomes. In 11 of these genomes, from phylogenetically distant bacteria, the FN3K homologue was immediately preceded by a low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homologue, which is therefore probably functionally related to the FN3K homologue. Five bacterial FN3K homologues (from Escherichia coli, Enterococcus faecium, Lactobacillus plantarum, Staphylococcus aureus and Thermus thermophilus) were overexpressed in E. coli, purified and their kinetic properties investigated. Four were ribulosamine/erythrulosamine 3-kinases acting best on free lysine and cadaverine derivatives, but not on ribulosamines bound to the alpha amino group of amino acids. They also phosphorylated protein-bound ribulosamines or erythrulosamines, but not protein-bound fructosamines, therefore having properties similar to those of mammalian FN3K-related protein. The E. coli FN3K homologue (YniA) was inactive on all tested substrates. The LMW-PTP of T. thermophilus, which forms an operon with an FN3K homologue, and an LMW-PTP of S. aureus (PtpA) were overexpressed in E. coli, purified and shown to dephosphorylate not only protein tyrosine phosphates, but protein ribulosamine 5-phosphates as well as free ribuloselysine 5-phosphate and erythruloselysine 4-phosphate. These LMW-PTPs were devoid of ribulosamine 3-phosphatase activity. It is concluded that most bacterial FN3K homologues are ribulosamine/erythrulosamine 3-kinases. They may serve, in conjunction with a phosphatase, to deglycate products of glycation formed from ribose 5-phosphate or erythrose 4-phosphate