Estudios estructurales de BRMS1 y Tax1BP1, proteínas involucradas en la vía de activación de NFkB

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-03-0

BRMS1 51-98 and BRMS1 51-84 are crystal oligomeric coiled- coils with different stoichiometry, which behave as disordered proteins in solution

ABSTRACT The Breast cancer metastasis suppressor 1 gene suppresses metastasis without affecting the primary tumour growth. Cellular localisation of BRMS1 appears to be important for exerting its effects on metastasis inhibition. We recently described a nucleo-cytoplasmic shuttling for BRMS1 and identified a nuclear export signal within the N-terminal coiled coil. The structure of these regions shows an antiparallel coiled coil capable of oligomerising, which compromises the accessibility to the nuclear export signal consensus residues. We have studied the structural and biophysical features of this region to further understand the contribution of the N-terminal coiled coil to the biological function of BRMS1. We have observed that residues 85 to 98 might be important in defining the oligomerisation state of the BRMS1 N-terminal coiled coil. In addition, we report the presence of a conformational dynamic equilibrium (oligomeric folded species ↔ oligomeric unfolded) in solution in the BRMS1 N-terminal coiled coil that might facilitate the nuclear export of BRMS1 to the cytoplasm.

Plastic degradation by insect hexamerins: Near-atomic resolution structures of the polyethylene-degrading proteins from the wax worm saliva

14 p.-8 fig.Plastic waste management is a pressing ecological, social, and economic challenge. The saliva of the lepidopteran Galleria mellonella larvae is capable of oxidizing and depolymerizing polyethylene in hours at room temperature. Here, we analyze by cryo–electron microscopy (cryo-EM) G. mellonella’s saliva directly from the native source. The three-dimensional reconstructions reveal that the buccal secretion is mainly composed of four hexamerins belonging to the hemocyanin/phenoloxidase family, renamed Demetra, Cibeles, Ceres, and a previously unidentified factor termed Cora. Functional assays show that this factor, as its counterparts Demetra and Ceres, is also able to oxidize and degrade polyethylene. The cryo-EM data and the x-ray analysis from purified fractions show that they self-assemble primarily into three macromolecular complexes with striking structural differences that likely modulate their activity. Overall, these results establish the ground to further explore the hexamerins’ functionalities, their role in vivo, and their eventual biotechnological application.This work was funded by Roechling Stiftung to F.B., Consejo Superior de Investigaciones Cientificas (CSIC) to F.B., Ministerio de Ciencia e Innovación (grants PID2019-111215RB-100 and PDC2022-133955-I00) to T.T., Ministerio de Ciencia e Innovación (grant PID2019-111215RB-100) to T.T., the Generalitat de Catalunya (2017 SGR 1192) to M.S., the Spanish Ministry of Science, Innovation and Universities MCIN/AEI/10.13039/501100011033 ERDF “A way to make Europe” PID2021-129038NB-I00 to M.S., and MCIN/AEI/10.13039/501100011033 (grant PID2020-120275GB-I00) to E.A.-P.Peer reviewe

Wax worm saliva and the enzymes therein are the key to polyethylene degradation by Galleria mellonella

11 p.-6 fig.Plastic degradation by biological systems with re-utilization of the by-products can be the future solution to the global threat of plastic waste accumulation. We report that the saliva of Galleria mellonella larvae (wax worms) is capable of oxidizing and depolymerizing polyethylene (PE), one of the most produced and sturdy polyolefin-derived plastics. This effect is achieved after a few hours’ exposure at room temperature and physiological conditions (neutral pH). The wax worm saliva can indeed overcome the bottleneck step in PE biodegradation, that is the initial oxidation step. Within the saliva, we identified two enzymes that can reproduce the same effect. This is the first report of enzymes with this capability, opening up the way to new ground-breaking solutions for plastic waste management through bio-recycling/up-cycling.Roechling Stiftung to FB Consejo Superior de Investigaciones Científicas (CSIC) to FB NATO Science for Peace and Security Programme (Grant SPS G5536) to TT Junta de Castilla y León, Consejería de Educación y Cultura y Fondo Social Europeo (Grant BU263P18) to TT Ministerio de Ciencia e Innovación (Grant PID2019-111215RB-100) to TT The Generalitat de Catalunya (2017 SGR 1192) to MS Ministerio de Ciencia e Innovación (Grant BFU2017-89143-P) to EA-PN

Crystallization and preliminary X-ray diffraction analysis of a breast cancer metastasis suppressor 1 predicted coiled-coil region

Crystals of human breast cancer metastasis Suppressor 1 N-terminal coiled-coil have been obtained and diffracted to 2.0 Å

Human Drg1 is a potassium-dependent GTPase enhanced by Lerepo4

11 páginas, 5 figuras, 1 tabla. Contiene 5 figuras en material suplementario, en este fichero.Human Drg1, a guanine nucleotide binding protein conserved in archaea and eukaryotes, is regulated by Lerepo4. Together they form a complex which interacts with translating ribosomes. Here we have purified and characterized the GTPase activity of Drg1 and three variants, a shortened mutant depleted of the TGS domain, a phosphomimicking mutant and a construct with the two combined mutations. Our data reveal that potassium strongly stimulates the GTPase activity, without changing the monomeric status of Drg1 and that this activity is notably reduced in the mutants. The nature of Lerepo4 association has also been investigated. Dissecting the role of the different domains revealed that Dfrp domain is the sole responsible for the Drg1 increase in thermal stability and the four fold stimulation over its catalytic activity. Lerepo4 action leaves Drg1 affinity for nucleotides unaffected, feasibly favoring a switch I reorientation, mainly via the TGS domain. Drg1 displayed a high temperature optimum of activity at 42°C, suggesting the ability of being active under possible heat stress conditions.This work was supported by Ministerio de Ciencia e Innovación (SAF2009-10667, SAF2012-31405) and Generalitat Valenciana (Prometeo/2012/061), Spain.Peer reviewe

Crystal structure of Hcp from Acinetobacter baumannii: a component of the type VI secretion system

16 p.-6 fig.-1 tab.The type VI secretion system (T6SS) is a bacterial macromolecular machine widely distributed in Gram-negative bacteria, which transports effector proteins into eukaryotic host cells or other bacteria. Membrane complexes and a central tubular structure, which resembles the tail of contractile bacteriophages, compose the T6SS. One of the proteins forming this tube is the hemolysin co-regulated protein (Hcp), which acts as virulence factor, as transporter of effectors and as a chaperone. In this study, we present the structure of Hcp from Acinetobacter baumannii, together with functional and oligomerization studies. The structure of this protein exhibits a tight β barrel formed by two β sheets and flanked at one side by a short α-helix. Six Hcp molecules associate to form a donut-shaped hexamer, as observed in both the crystal structure and solution. These results emphasize the importance of this oligomerization state in this family of proteins, despite the low similarity of sequence among them. The structure presented in this study is the first one for a protein forming part of a functional T6SS from A. baumannii. These results will help us to understand the mechanism and function of this secretion system in this opportunistic nosocomial pathogen.Peer reviewe

The structure of BRMS1 nuclear export signal and SNX6 interacting region reveals a hexamer formed by antiparallel coiled coils

14 páginas, 3 tablas, 3 figuras. PMID:21777593[PubMed] Coordinates and structure factors have been deposited in the PDB with accession number 2XUSWe present here the first structural report derived from breast cancer metastasis suppressor 1 (BRMS1), a member of the metastasis suppressor protein group, which, during recent years, have drawn much attention since they suppress metastasis without affecting the growth of the primary tumor. The relevance of the predicted N-terminal coiled coil on the molecular recognition of some of the BRMS1 partners, on its cellular localization and on the role of BRMS1 biological functions such as transcriptional repression prompted us to characterize its three-dimensional structure by X-ray crystallography. The structure of BRMS1 N-terminal region reveals that residues 51-98 form an antiparallel coiled-coil motif and, also, that it has the capability of homo-oligomerizing in a hexameric conformation by forming a trimer of coiled-coil dimers. We have also performed hydrodynamic experiments that strongly supported the prevalence in solution of this quaternary structure for BRMS1(51-98). This work explores the structural features of BRMS1 N-terminal region to help clarify the role of this area in the context of the full-length protein. Our crystallographic and biophysical results suggest that the biological function of BRMS1 may be affected by its ability to promote molecular clustering through its N-terminal coiled-coil region.This work was supported by Ministerio de Ciencia e Innovación (SAF2008-04048-E and SAF2009-10667); Conselleria de Sanitat, Generalitat Valenciana (AP-001/10); Consejo Superior de Investigaciones Científicas (200820I020); and Fundación Mutua Madrileña, Spain. Work in JLN laboratory was supported by Ministerio de Ciencia e Innovacion (CSD2008-00005, SAF2008-05742-C2-01), Generalitat Valenciana (ACOMP2011/113) and Fundación para la investigación y prevención del SIDA en España, FIPSE, (36557/06).Peer reviewe

Biochemical and structural characterization of a widespread family of transposons

2 p.-1 fig.Peer reviewe

Biochemical and structural characterization of a widespread family of transposons

Transposition is the translocation of a DNA fragment, from a donor site to a target site showing no sequence homology. These mobile DNAs, called transposons, are guided by nucleoprotein complexes to mediate transposition events. The transposases are enzymes that bind to the end of the transposon and catalyze its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism [1]. Transposases are among the most prevalent genes in nature, and their DNA reshuffling activity can modify gene expression, promote evolution of species, and spread antibiotic resistance and virulence factors. Because of this, transposons have been widely associated with different human diseases. Importantly, moreover, some of these mobile elements are regularly used as biotechnological tools. However, the molecular mechanisms that control DNA transposition remain poorly understood. Here we focus on IS21, one of the most widespread families of transposons. Notably, these streamlined mobile elements (Figure 1) contain two transposon ends, termed Terminal Inverted Repeats (TIRs), which are recognized and processed by the transposase [2]. Between them, there are two proteins coding genes: the canonical DDE transposase (IstA) and, in a similar way to some classic transposons such as Mu or Tn7,an essential regulatory ATPase that belongs to the AAA+ super-family of proteins (IstB) [3]. We have used cryo-electron microscopy, in combination with biochemical assays, to analyse the molecular mechanisms and regulation of IS21. In particular, we have characterized the molecular determinants of the transposon ends, how the transposase recognizes these sequences and how they, in turn, modulate IstA function. Overall, our results provide fundamental insights into IS21 transposition and reveal how IstA specifically recognizes the transposon ends to prevent chromosomal breaks and promote efficient DNA transposition.The authors acknowledge funding from the Spanish Ministry of Science and Innovation, the Spanish Research Agency, the European Social Fund and the European Regional Development Fund.Peer reviewe