Identification of the first structurally validated covalent ligands of the small GTPase RAB27A

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein–protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket (‘WF-binding pocket’) via a conserved tryptophan–phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets

Projecte científico tècnic de definició de subunitats paisatgístiques del Parc Natural de l'Alt Pirineu

La identificació i caracterització de subunitats de paisatge per al Parc Natural de l'Alt Pirineu (PNAP) s'ha fet en base a la Llei 8/2005, de 8 de juny, de Protecció, Gestió i Ordenació del Paisatge la qual estableix que "els catàlegs del paisatge són els documents de carácter descriptiu i prospectiu que determinen la tipologia dels paisatges de Catalunya, identifiquen llurs valors i llur estat de conservació i proposen els objectius de qualitat que han de complir" (Article 10). Igualment, la Llei ha previst que la responsabilitat dels catàlegs sigui de l'Observatori del Paisatge que "és una entitat de suport i col·laboració amb l'Administració de la Generalitat en totes les qüestions relacionades amb l'elaboració, l'aplicació i la gestió de les polítiques de paisatge" (Article 13). Per això, aquest treball es basarà en els criteris establerts per l'Observatori del Paisatge (document de referència pels grups de treball, Olot i Barcelona, maig de 2005) i les bases conceptuals, metodològiques i procedimentals que han elaborat per a la realització dels Catàlegs del Paisatge de Catalunya que s'han recollit en un document anomenat Prototipus de Catàleg de Paisatge (PCP). Com que en l'actualitat aquests paràmetres estan en fase de desenvolupament, aquest Projecte Científico Tècnic de Definició de Subunitats Paisatgístiques per al Parc Natural de l'Alt Pirineu s'ha inspirat en aquest document adaptant-se a les necessitats del Parc i desenvolupant les metodologies proposades. El document que es presenta recull la Primera Fase del treball, que s'ha destinat al tractament i adequació de la informació existent (cartogràfica i escrita) per a la delimitació i definició posterior de les diferents subunitats de paisatge

Palaeoenvironmental changes in the Iberian central system during the Late-glacial and Holocene as inferred from geochemical data: A case study of the Navamuño depression in western Spain

The Iberian Central System (ICS) is a clue region to reveal Mediterranean/Atlantic inferences over Iberia. We present a multidisciplinary study from western Spain conducted in the Navamuno ˜ depression (ND), covering the last 16.8 ka (cal BP). A reconstruction of the palaeotemperature from the resulting geochemical data highlights four cold and dry intervals, namely, the Oldest Dryas, Older Dryas, Intra-Allerød Cold Period (IACP), and the Younger Dryas, along with warmer intervals: the Bølling (14.7–14 ka) and the Allerød (12.9–12.6 ka); however, the Greenland Interstadial GI-1c (13.4–13.1 ka) is barely distinguishable in the ND. Despite the shortage of biomass to sustain fire, the earliest charcoals are from ~14.4–13.8 ka. Evidence of ash/dust events overprinting the geochemical background starts at ~13.8–12.8 ka. Significant fire activity in the Early Holocene at ~11.7–10.6 ka affected the ND, matching the westernmost ICS data. This period includes short oceanic spells inferred from Cl peaks at ~10.9–10.2 ka and three cold intervals at 11.4, 9.3, and 8.2 ka disrupted the progressive temperature increase. The Mid-Holocene showed a continuously increasing trend towards an arid climate, peaking at 4.2 ka under a pervasive dust influx from North Africa, which has prevailed since almost ~7.9 ka. A prominent volcanic event at ~6.8–5.8 ka is in Navamuno ˜ and Ronanzas ˜ (Asturias, N Spain; Gallego et al., 2013) identified from heavy metal-rich layer, synchronous with the last known eruption of the Calatrava volcanic field (South-Central Spain; Poblete-Piedrabuena et al., 2019). This volcanic eruption could affect many other regions half north of Iberia. The pervasive presence of oceanic aerosols in the last three millennia (2.8 ka ~ ) allowed the formation of a Cl-rich peat layer during the Ibero-Roman humid period ~2.1 ka, before a changing around ~0.4 ka toward colder and drier conditions at the Little Ice Age (LIA) periodThis study was funded by the LATESICE-CGL2016-78380-P, FINICES- PID2020-117685 GB-I00 and MED-REFUGIA-RTI2018-101714-BI00 (Plan Nacional I + D + I, Spanish Ministry of Science, Innovation and Universities) projects

Paleoenvironmental Reconstruction for the Last 3500 Years in the Southern Pyrenees from a Peat Bog Core in Clots de Rialba

Vegetation landscape dynamics are derived from the relationships established between anthropic activities and climate conditions over time. Paleoenvironmental research in the Pyrenees range (north-eastern Iberian Peninsula) has revealed what these dynamics were like in some regions during the Holocene. However, some fields of biogeography still present questions that need to be addressed, such as the patterns of Fagus sylvatica and Abies alba and the importance of the fire regime during the Meghalayan (late Holocene). We present a multiproxy study performed in a sedimentary record from the Clots de Rialba peat bog, located at 2093 m a. s. l. (Lleida, southern slope of the Pyrenees mountain range), that covers the last c. 3500 years. Analyses were performed on the organic matter content, pollen, non-pollen palynomorphs, and sedimentary charcoals larger than 150 μm. The palynological spectra revealed a maximum extension of Abies alba at about 3500 cal yr BP in the Bronze Age, while Fagus sylvatica showed its maximum extent between 3300 and 2800 cal yr BP. A dominance of Pinus was detected throughout the studied period. Other taxa such as cereals, herbaceous plants, aquatic plants, and coprophilous fungi have also been discussed to identify anthropic pressure and climate pulses. In addition, the study of sedimentary charcoals reveals the main forest fire episodes and their recurrences, some of them linked to anthropogenic activities and/or climate variations. These anthropogenic activities would include the use of opening and maintaining forest fires in deforestation in order to obtain pastures and spaces dedicated to cereal agriculture and the appearance of some taxa linked to or introduced by human communities

Molecular ruler mechanism and interfacial catalysis of the integral membrane acyltransferase PatA

Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity “ruler.” We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acyl–coenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors

Rv2466c mediates the activation of TP053 to kill replicating and non-replicating Mycobacterium tuberculosis.

The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053, and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c, demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7Å resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a β-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti-mycobacterial activity of TP-based compounds paving the way for future drug development programmes