Biology subjects

Pellegrini, E.

Publications and source records attributed to Pellegrini, E..

7 recordsLinked to original sources

bioRxiv2025

Variations in the root-soil system influence the grapevine holobiont by shaping plant physiology and root microbiome

Soil-dwelling bacteria and fungi play a crucial role in plant health and productivity by engaging in complex interactions that shape and are shaped by soil physico-chemical properties. In this study, we employed a multi-omics approach to investigate how variations in soil composition affect the grapevine holobiont. Grape plantlets were grown in three distinct soil types, namely sand, peat, and peat-manure. To further assess how variation in soil and root conditions affects the holobionts response, we included treatments involving soil autoclaving and root surface sterilisation across all soil types. We found that soil type significantly influences leaf multielement composition and concentration, while also shaping the bacterial and fungal communities associated with the plant rhizosphere. This shift led to changes in taxa involved in nitrogen fixation, biocontrol, and pathogenicity. Autoclaving soils consistently reduced bacterial diversity across all soil types, whereas fungal communities were less affected. In contrast, thermal treatment of roots had only a minor impact on microbial community composition but did induce transcriptional changes in the root and altered leaf macronutrient concentrations. Our findings indicate that differences in soil composition reshape the entire root-soil continuum, ultimately affecting plant physiology at multiple levels--from root function to leaf nutrient status. This highlights that the soil is not a passive growth medium but a key determinant of grape holobiont structure and function. These results reinforce the view that plant health and adaptation arise from integrated, dynamic interactions among the host, its associated microbiome, and the surrounding soil matrix.

plant biology↗

bioRxiv2024

Metal fluorides - multi-functional tools for the study of phosphoryl transfer enzymes

Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein group across all kingdoms of life, making up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyse these reactions is essential in characterising the processes they regulate. Metal fluorides can be used as multifunctional tools for the study of these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogues. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryoEM and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal with soft X-rays. Highlights Enzymes that catalyse phosphoryl transfer are the largest family of enzymes and are involved in the storage and transmission of genetic information, energy transfer, signalling and cellular differentiation Metal fluorides form a comprehensive tool kit to study the mechanisms of these enzymes by stabilizing the active conformation; mimicking both the transition state and ground state; and placing spectroscopically sensitive atoms into the active site A guide is presented to the optimal formation of these complexes and their use in a wide variety of techniques in structural biology

molecular biology↗

bioRxiv2024

The MAP2Ks can use ADP to phosphorylate and activate their substrate MAPKs in vitro

Kinases are a diverse group of enzymes that use ATP to phosphorylate a variety of substrates. Protein kinases evolved in eukaryotes as important mediators of cell signalling that target specific amino acid side chains to modulate downstream protein function. Among them, the MAPKs (mitogen-activated protein kinases) are a family of intracellular protein kinases that form signalling cascades responding to a number of stimuli, that control fundamental mechanisms such as proliferation, differentiation, inflammation and cell death. Signals propagate through consecutive kinases which eventually phosphorylate and activate a MAPK. Here, we show that the dual specificity threonine/tyrosine MAP kinase kinases (MAP2Ks or MEKs) are able to phosphorylate and activate their substrate MAPKs using ADP as well as ATP in vitro. As the pathways are involved in the stress response, we speculate that it would represent an advantage to be able to maintain signalling under conditions such as hypoxia, that occur under a number of cell stresses, including cancer and atherosclerosis, where the available pool of ATP could be depleted.

biochemistry↗

bioRxiv2023

Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex

The nuclear cap-binding complex (CBC) co-ordinates co-transcriptional maturation, transport, or degradation of nascent Pol II transcripts. CBC with its partner ARS2 form mutually exclusive complexes with diverse effectors that promote either productive or destructive outcomes. Combining Alphafold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18 and other effectors compete for binding to ARS2. In ternary CBCA complexes with either PHAX, NCBP3 or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that in vivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.

biochemistry↗

bioRxiv2022

Structural analysis shows that the BIR2 domain of E3 ligase XIAP binds across the RIP2 kinase dimer interface

NOD1 and NOD2 are innate immune system pattern recognition receptors that play a key role in the protection from bacterial infections and in the maintenance of gastro-intestinal homeostasis. Dysregulation of NOD signalling pathways promotes chronic inflammatory diseases such as inflammatory bowel disease. RIP2, which contains a kinase domain (RIP2K) and a CARD domain, is the immediate downstream signalling partner in the NOD pathway and inhibition of its kinase activity and ubiquitination are promising strategies to address these inflammatory diseases. However, recent work indicates that the phosphorylation activity of RIP2K is dispensable for signalling and that inhibitors of both RIP2K activity and RIP2 ubiquitination prevent the essential interaction between RIP2K and the BIR2 domain of XIAP, the key RIP2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined Native mass spectrometry, NMR, cryo-electron microscopy and Alphafold2 predictions to determine the structure of the RIP2K-XIAP BIR2 complex with 2:1 stoichiometry. The structure shows that complex formation requires that the kinase is in an active-like (i.e. C-helix IN) and dimeric conformation and explains both inhibitory mechanisms. It also shows why phosphorylation of the kinase activation loop is dispensable for signalling and reveals the structural role of RIP2K-K209 residue in the RIP2K-XIAP BIR2 interaction. Importantly, our results clarify the features of the RIP2K conformation essential for its role as a scaffold protein for ubiquitination.

biochemistry↗

bioRxiv2022

Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation

The MAP kinase p38 is a central component of signalling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double-phosphorylation from MAP2Ks, due to the challenge of trapping a transient and dynamic hetero-kinase complex. Here, we applied a multidisciplinary approach to generate the first structure of p38 in complex with its MAP2K MKK6 and understand the activation mechanism. Integrating cryo-EM with MD simulations, HDX-MS and in cellulo experiments, we demonstrate a dynamic, multi-step, phosphorylation mechanism, reveal new catalytically relevant interactions, and show that MAP2K disordered N-termini determine pathway specificity. Our work captures, for the first time, a fundamental step of cell signalling: a kinase phosphorylating its downstream target kinase. One-Sentence SummaryIntegrative Cryo-EM and MD analysis of an active hetero-kinase complex reveals details of cellular signal transmission

biophysics↗

bioRxiv2019

Quantitative Measurements of Enlarged Perivascular Spaces in the Brain are Associated with Retinal Microvascular Parameters in Older Community-Dwelling Subjects

BackgroundPerivascular Spaces (PVS) become increasingly visible with advancing age on brain MRI, yet their relationship to morphological changes in the underlying microvessels remains poorly understood. Retinal and cerebral microvessels share morphological and physiological properties. We compared computationally-derived PVS morphologies with retinal vessel morphologies in older people. MethodsWe analysed data from community-dwelling individuals who underwent multimodal brain MRI and retinal fundus camera imaging at mean age 72.55 years (SD=0.71). We assessed centrum semiovale PVS computationally to determine PVS total volume and count, and mean per-subject individual PVS length, width and size. We analysed retinal images using the VAMPIRE software suite, obtaining the Central Retinal Artery and Vein Equivalents (CRVE and CRAE), Arteriole-to-Venule ratio (AVR), and fractal dimension (FD) of both eyes. We investigated associations using general linear models, adjusted for age, gender, and major vascular risk factors. ResultsIn 381 subjects with all measures, increasing total PVS volume and count were associated with decreased CRAE in the left eye (volume {beta}=-0.170, count {beta}=-0.184, p<0.001). No associations of PVS with CRVE were found. The PVS total volume, individual width and size increased with decreasing FD of the arterioles (a) and venules (v) of the left eye (total volume: FDa {beta}=-0.137, FDv {beta}=-0.139, p<0.01; width: FDa {beta}=-0.144, FDv {beta}=-0.158, p<0.01; size: FDa {beta}=-0.157, FDv {beta}=-0.162, p<0.01). ConclusionsIncrease in PVS number and size visible on MRI reflect arteriolar narrowing and lower retinal arteriole and venule branching complexity, both markers of impaired microvascular health. Computationally-derived PVS metrics may be an early indicator of failing vascular health and should be tested in longitudinal studies.

neuroscience↗