Baculovirus expression:old dog, new tricks

International audienc

Use of an innovative system and nanotechnology-based strategy for therapeutic applications of Gla-rich protein (GRP)

Introduction: Gla-rich protein (GRP) is a vitamin K-dependent protein (VKDP) acting as a calcification inhibitor and anti-inflammatory agent in cardiovascular and articular systems, and THP1 monocyte/macrophage cells [1,2]. Calcification and inflammation processes are known to be involved in the etiology of several calcification-related chronic inflammatory diseases such as atherosclerosis, CKD and osteoarthritis, in a complex bi-directional interplay that drives disease progression. Here, we developed an innovative system to produce human c-carboxylated GRP (cGRP), and a nanotechnology strategy based on GRP loading into extracellular vesicles (EVs) as a gold standard delivery system for GRP in therapeutic applications. Materials and methods: Human GRP protein was co-expressed with c-carboxylase enzyme (GGCX), vitamin K oxidoreductase (GGCX) and furin, in the insect cell baculovirus system in the presence of vitamin K. GRP released in the cell culture media was characterized by mass spectrometry based techniques and Western blot analysis. EVs released by the insect cells overexpressing GRP were isolated by ultracentrifugation, and characterized for GRP content through TEM-immunogold staining, Western blot, ELISA, qPCR. Functional assays using isolated EVs containing GRP were performed in primary vascular smooth muscle cells (VSMCs) and THP1 monocyte/macrophage cells, for anti-mineralizing and anti-inflammatory screening.Results: GRP released in the cell culture media when co-expressed with GGCX, VKOR and furin in the presence of vitamin K, is processed at the pro-peptide and contain Gla residues. EVs released by the insect cells in this system were shown to be loaded with GRP protein and mRNA, and capable of reducing ECM calcium deposition of calcifying VSMCs and the production of TNFa in THP1 monocyte/macrophage cells stimulated with LPS. Discussion and conclusions: While the successful production of human cGRP constitutes a major achievement, this innovative methodology will open new opportunities for the production of other biological active VKDPs. Furthermore, EVs loaded with GRP were shown to have anti-mineralizing and anti-inflammatory properties, with promising therapeutic potentialities for calcification-related chronic inflammatory diseases.Portuguese Foundation for Science and Technology (EU/PID1003201)info:eu-repo/semantics/publishedVersio

Sequence, overproduction and crystallization of aspartyl-tRNA synthetase from Thermus thermophilus Implications for the structure of prokaryotic aspartyl-tRNA synthetases

AbstractThe genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermophilus strains VK.-1 and HB8, have been cloned and sequenced. Their nucleotidic sequences code for the same protein which displays the three characteristic motifs of class II aminoacyl-tRNA synthetases. This enzyme shows 50% identity with Escherichia coli AspRS, over the totality of the chain (580 amino acids). A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of an extra domain between motifs 2 and 3 much larger than in the eukaryotic ones. When its gene is under the control of the tac promoter of the expression vector pKK223-3, the protein is efficiently overexpressed as a thermostable protein in E. coli. It can be further purified to homogeneity using a heat treatment followed by a single anion exchange chromatography. Single crystals of the pure protein, diffracting at least to 2.2 Å resolution (space group P212121, a = 61.4 Å, b = 156.1 Å, c = 177.3 Å) are routinely obtained. The same crystals have previously been described as crystals of threonyl-tRNA synthetase [1]

PLoS Biol

The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription

ADN hélicases et maladies associées

Les hélicases sont des moteurs moléculaires utilisant l’énergie libérée par l’hydrolyse de l’ATP pour catalyser le remodelage des acides nucléiques, et notamment le déroulement de molécules d’ARN ou d’ADN double brin. De nombreuses hélicases participent au maintien de l’intégrité du génome, et leurs mutations peuvent entraîner des troubles du système de réparation avec des conséquences cliniques graves. Parmi les ADN hélicases dont les mutations sont à l’origine de maladies, on trouve notamment les hélicases de la famille RecQ, affectées dans les syndromes de Bloom, de Rothmund-Thomson et de Werner, l’hélicase BRIP1/BACH1, dont une déficience peut causer une anémie de Fanconi, ainsi que les sous-unités XPB et XPD du facteur de transcription/ réparation TFIIH, mutées chez des patients atteints de xeroderma pigmentosum, de trichothiodystrophie ou du syndrome de Cockayne

Initiation de la transcription et sa régulation (Vers une étude structurale des mécanismes de communication entre le facteur général de la transcription TFIIH et le récepteur nucléaire RAR)

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Etude de p8/TTD-A, une sous-unité de TFIIH impliquée dans la Trichothiodystrophie de type A (Structure et interactions avec ses partenaires)

Le facteur TFIIH est un complexe multi-protéique composé de 10 sous-unités, impliqué dans l initiation de la transcription ainsi que dans la réparation de l ADN par excision resynthèse. Des mutations dans les gènes codant pour trois de ses sous-unités sont à l origine de trois maladies héréditaires caractérisées par des défauts de réparation et/ou transcription. Je me suis focalisé sur la sous-unité p8, dont une absence ou un dysfonctionnement sont à l'origine de la trichothiodystrophie de groupe A. J ai déterminé la structure de p8 par RMN et étudié avec diverses approches biophysiques des complexes entre p8 et p52, son partenaire dans le complexe TFIIH. L interprétation de ces données, à la lumière de la structure cristalline du complexe p8-p52 de levure, fourni une explication moléculaire des effets déstabilisateurs des mutations observées chez les malades. J ai également participé à la conception d une nouvelle méthode d enregistrement de données de relaxation par RMN, permettant l accès au temps de corrélation de rotation d une protéine en solution avec un gain de temps d un facteur quatre par rapport aux enregistrements classiques.TFIIH, a multi-subunit complex composed of 10 subunits, is important for transcription initiation and for DNA nucleotide excision repair. Mutations in the genes coding for three TFIIH subunits cause hereditary diseases characterized by defects in repair and/or transcription. I focused on p8, a subunit whose absence or dysfunction cause type A trichothiodystrophy. I determined the structure of p8 by NMR and studied, with diverse biophysical approaches, complexes between p8 and p52, its partner within TFIIH. Interpretation of these data, in the light of yeast crystal structure of p8-p52 complex, provides a molecular explanation of the destabilizing effects caused by the mutations observed in patients. I also participated in the conception of a novel method for recording NMR relaxation data, allowing the rotational correlation time of a protein to be determined in a time four times shorter than classical experiments.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF