Biology subjects

Acuto, O.

Publications and source records attributed to Acuto, O..

8 recordsLinked to original sources

bioRxiv2026

Molecular determinants of differential substrate selection between the Src family kinases Lck and Src

Src family kinases (SFKs) share highly conserved catalytic domains yet display distinct biological functions, raising the question of how substrate specificity is achieved. Here, we investigate the molecular basis of differential ITAM recognition by Lck and Src, combining cellular assays with structural analysis and docking simulations. In-cell assays demonstrated that, contrary to Lck, Src was completely incapable of phosphorylating the TCR ITAMs when ectopically expressed in a T cell environment. Domain-swapping experiments further revealed that substitution of the Src kinase domain with that of Lck was sufficient to confer ITAM phosphorylation and trigger downstream TCR signaling responses, whereas exchange of adaptor domains had minimal effect. Comparative structural analysis revealed that, despite their overall conserved fold, Lck exhibits a more open and solvent accessible pocket located between the N- and C-lobes of the kinase domain, adjacent to the activation loop, compared to Src. Consistent with this, docking simulations showed that Lck accommodates ITAM peptides in multiple favourable conformations, whereas Src displays a markedly reduced number of non-productive binding poses. Residue-level contact analysis identified a defined interaction surface in Lck, spanning the inter-lobal regions and activation loop. Our results highlight the importance of kinase domain conformational landscape in shaping substrate selectivity and have implications for the rational design of selective SFK inhibitors.

Cell Biology↗

bioRxiv2022

Molecular dynamics simulations reveal membrane lipid interactions of the full-length lymphocyte specific kinase Lck

The membrane-bound lymphocyte-specific protein-tyrosine kinase (Lck) triggers T cell antigen receptor signalling to initiate adaptive immune responses. Despite many structure-function studies, the mode of action of Lck and the potential role of plasma membrane lipids in regulating Lcks activity remains elusive. Advances in molecular dynamics simulations of membrane proteins in complex lipid bilayers have opened a new perspective in gathering such information. Here, we have modelled the full-length Lck open and closed conformations available from crystallographic studies and simulated its interaction with the inner leaflet of the T cell plasma membrane. In both conformations, we found that the unstructured unique domain and the structured domains including the kinase interacted with the membrane with a preference for PIP lipids. Interestingly, our simulations suggest that the Lck-SH2 domain interacts with lipids differently in the open and closed Lck conformations, demonstrating that lipid interaction can potentially regulate Lcks conformation and in turn modulate T cell signalling. Additionally, the Lck-SH2 and kinase domain residues that significantly contacted PIP lipids are found to be conserved among the Src family of kinases, thereby potentially representing similar PIP interactions within the family.

biophysics↗

bioRxiv2022

A role of Lck annular lipids in the steady upkeep of active Lck in T cells

Theoretical work suggests that collective spatiotemporal behaviour of integral membrane proteins (IMPs) can be modulated by annular lipids sheathing their hydrophobic moiety. Here, we present evidence for this prediction in a natural membrane by investigating the mechanism that maintains steady amount of active isoform of Lck kinase (LckA) by Lck trans-autophosphorylation offset by the phosphatase CD45. We gauged experimental suitability by quantitation of CD45 and LckA subcellular localisation, LckA generation as a function of Lck and pharmacological perturbation. Steady LckA was challenged by swapping Lck membrane anchor with structurally divergent ones expected to substantially modify Lck annular lipids, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45, respectively. The data showed only small alteration of LckA, except for CD45 hydrophobic anchor that thwarted LckA, due to excessive lateral proximity to CD45. The data are best explained by annular lipids facilitating or penalising IMPs lateral proximity, hence modulating IMPs protein-protein functional interactions. Our findings can contribute to improve the understanding of biomembranes organisation.

cell biology↗

bioRxiv2021

Multi-scale simulations of the T cell receptor reveal its lipid interactions, dynamics and the arrangement of its cytoplasmic region

The T cell antigen receptor (TCR-CD3) complex initiates T cell activation following recognition of peptides presented by Major Histocompatibility Complex (pMHC)-encoded proteins. The ligation of pMHC to TCR{beta} induces Src family kinases activity via the cytoplasmic tails of the CD3{delta}{varepsilon}, CD3{gamma}{varepsilon} and {zeta}{zeta} dimers. The TCR-CD3 topology is well understood, but little is known about its conformational dynamics and arrangement of its cytoplasmic tails, limiting our grasp of the signalling mechanism. Here, we investigated the entire TCR-CD3 embedded in an asymmetric lipid bilayer using molecular modelling and multi-scale molecular dynamics simulations. Our study demonstrates conformational changes in the extracellular and transmembrane domains, and the arrangement of the TCR-CD3 cytoplasmic tails. The TCR{beta} variable regions were the most flexible in the extracellular domain. The cytoplasmic tails formed highly interlaced structures while some tyrosine sidechains within the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3{varepsilon} and {zeta} subunits dynamically penetrated the hydrophobic core of the bilayer. Ionic interactions between the cytoplasmic tails and phosphatidylinositol phosphates (PIP2 and PIP3) in the inner leaflet of the lipid bilayer led to the formation of a distinct annular lipid fingerprint around the TCR-CD3 complex. These results combined with available experiential data increase our understanding of the TCR-CD3 activation mechanism and highlight the importance of membrane lipids in regulating T cell activation. Significance statementThe T cell receptor (TCR-CD3) detects antigenic peptides displayed by major histocompatibility complexes (pMHC) to instigate activation of T cell adaptive immunity. Despite significant structural and functional knowledge of TCR-CD3 topology, the membrane interactions and dynamics of its cytoplasmic moieties remain elusive. Interactions of TCR-CD3 cytoplasmic tails with membrane lipids may regulate their phosphorylation by Src-family kinases, the first intracellular event required for T cell activation. Using the static 3D structure of TCR-CD3 resolved by cryo-electron microscopy, we provide novel insights into the protein-lipid interactions of the complete TCR-CD3 embedded in a bilayer closely mimicking its native membrane environment. Our study sheds light on the dynamics of the TCR-CD3 at near-atomic resolution and further aids in deciphering its activation mechanism.

biophysics↗

bioRxiv2020

Allosteric activation of T cell antigen receptor signalling by quaternary structure relaxation

The mechanism of T cell antigen receptor (TCR-CD3) signalling remains elusive. Here, we identified mutations in the transmembrane region of TCR{beta} or CD3{zeta} that augmented pMHC-induced signalling, not explicable by enhanced ligand binding, lateral diffusion, clustering or co-receptor function. Using a novel biochemical assay and molecular dynamics simulation, we demonstrated that the gain-of-function mutations loosened interaction between TCR{beta} and CD3{zeta}. We found that, similar to the activating mutations, pMHC binding reduced TCR{beta} cohesion with CD3{zeta}. This event occurred prior to CD3{zeta} phosphorylation and at 0{degrees}C. Moreover, we demonstrated that soluble monovalent pMHC alone induced signalling and reduced TCR{beta} cohesion with CD3{zeta} in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCR{beta} to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to licence CD3{zeta} phosphorylation and initiate signal propagation.

immunology↗

bioRxiv2020

The full model of the pMHC-TCR-CD3 complex: a structural and kinetics characterization

The machinery involved in cytotoxic T-cell activation requires three main characters such as: the major histocompatibility complex class I (MHC I) bound to the peptide (p), the T-cell receptor (TCR), and the CD3-complex which is a multidimer interfaced with the intracellular side. The pMHC:TCR interaction has been largely studied both in experimental and computational models, giving a contribution in understanding the complexity of the TCR triggering process. Nevertheless, a detailed study of the structural and dynamical characterization of the full complex (pMHC:TCR:CD3-complex) is still missing, due to insufficient data available on the CD3-chains arrangement around the TCR. The recent determination of the TCR:CD3-complex structure by means of Cryo-EM technique has given a chance to build the entire proteins system essential in the activation of T-cell, and thus in the adaptive immune response. Here, we present the first full model of the pMHC interacting with the TCR:CD3-complex, built in a lipid environment. To describe the conformational behaviour associated with the unbound and the bound states, all atoms Molecular Dynamics simulations were performed for the TCR:CD3-complex and for two pMHC:TCR:CD3-complex systems, bound to two different peptides. Our data point out that a conformational change affecting the TCR Constant {beta} (C{beta}) region occurs after the binding to the pMHC, revealing a key role of such a region in the propagation of the signal. Moreover, we found that the TCR reduces the flexibility of the MHC I binding groove, confirming our previous results.

immunology↗

bioRxiv2020

Activation of Src-family kinases orchestrate secretion of flaviviruses by targeting mature progeny virions to secretory autophagosomes

Among the various host cellular processes that are hijacked by flaviviruses, very few mechanisms have been described with regard to viral secretion. Here we investigated how flaviviruses exploit the Src family kinases (SFKs) for exit from infected cells. We isolated three members of the SFK family - Src, Fyn and Lyn - that were specifically activated during secretion of Dengue and Zika or their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs blocked virus secretion, most significantly upon Lyn-deficiency. Lyn-/- cells were severely impaired in virus release, and were rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We further established that Lyn, via its palmitoylation-dependent membrane association, triggered post-Golgi virus transport in specialised Rab11 and Transferrin receptor positive organelles resembling secretory autophagosomes, and distinct from conventional exocytic vesicles. In the absence of Lyn activity or its aberrant membrane association, virions were sorted into the lysosomal pathway for degradation. This mode of export was specifically triggered by processed, and mature, but not by furin-resistant virus particles, and occurred with significantly faster kinetics than the conventional secretory pathway. Our study therefore charts a previously undiscovered Lyn-dependent exit strategy, triggered by flaviviruses in secretory autophagosomes that might enable them to evade circulating antibodies and dictate tissue tropism.

microbiology↗

bioRxiv2019

Phosphoproteomics of CD2 signaling reveals an AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells

The in-depth analysis of costimulatory signaling enhancing the activity of cytotoxic T cells (CTLs) represents a major approach towards immunotherapy development. Here we report that CD2 costimulation plays a critical role in killing by freshly isolated human CTLs, which represent a challenging but valuable study model to gain insight into CTL biology. We show that CD2 triggering critically aids signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules towards the microtubule-organizing center (MTOC). To gain insight into the underlying elusive mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeleton organization, autophagy and metabolism. Strikingly, signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) represents a functionally critical node of the CD2 network which regulates granule polarization towards the MTOC in CTLs. Granule trafficking is driven by active AMPK enriched on adjacent lysosomes, illustrating a novel signaling cross-talk between vesicular compartments in CTLs. Our results thus establish CD2 signaling as key for regulating cytotoxic killing and granule polarization in freshly isolated CTLs and strengthens the rationale to choose CD2 and AMPK as therapeutic targets to boost CTL activity.

immunology↗