Susana Gordo

Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR–pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

Stability and structural recovery of the tetramerization domain of p53-R337H mutant induced by a designed templating ligand

Protein p53 is a transcription factor crucial for cell cycle and genome integrity. It is able to induce both cell arrest when DNA is damaged and the expression of DNA repair machinery. When the damage is irreversible, it triggers apoptosis. Indeed, the protein, which is a homotetramer, is mutated in most human cancers. For instance, the inherited mutation p53-R337H results in destabilization of the tetramer and, consequently, leads to an organism prone to tumor setup. We describe herein a rational designed molecule capable of holding together the four monomers of the mutated p53-R337H protein, recovering the tetramer integrity as in the wild-type structure. Two ligand molecules, based on a conical calix[4]arene with four cationic guanidiniomethyl groups at the wider edge (upper rim) and hydrophobic loops at the narrower edge (lower rim), fit nicely and cooperatively into the hydrophobic clefts between two of the monomers at each side of the protein and keep the tetrameric structure, like molecular templates, by both ion-pair and hydrophobic interactions. We found a good agreement between the structure of the complex and the nature of the interactions involved by a combination of theory (molecular dynamics) and experiments (circular dichroism, differential scanning calorimetry and 1H saturation transfer difference NMR).

Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain

The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide–MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.

Self-reactive human CD4 T cell clones form unusual immunological synapses.

Compared with influenza-specific T cells, self-reactive T cells from patients with multiple sclerosis or type 1 diabetes fail to slow down and do not form normal immunological synapses upon encounter with cognate self-peptide presented by MHC.

Knitting and untying the protein network: Modulation of protein ensembles as a therapeutic strategy

Proteins constitute the working machinery and structural support of all organisms. In performing a given function, they must adopt highly specific structures that can change with their level of activity, often through the direct or indirect action of other proteins. Indeed, proteins typically function within an ensemble, rather than individually. Hence, they must be sufficiently flexible to interact with each other and execute diverse tasks. The discovery that errors within these groups can ultimately cause disease has led to a paradigm shift in drug discovery, from an emphasis on single protein targets to a holistic approach whereby entire ensembles are targeted.

Crossreactivity of a human autoimmune TCR is dominated by a single TCR loop.

Self-reactive CD4 T cells are thought to have a central role in the pathogenesis of many chronic inflammatory human diseases. Microbial peptides can activate self-reactive T cells, but the structural basis for such crossreactivity is not well understood. The Hy.1B11 T cell receptor (TCR) originates from a patient with multiple sclerosis and recognizes the self-antigen myelin basic protein. Here we report the structural mechanism of TCR crossreactivity with two distinct peptides from human pathogens. The structures show that a single TCR residue (CDR3α F95) makes the majority of contacts with the self-peptide and both microbial peptides (66.7-80.6%) due to a highly tilted TCR-binding topology on the peptide-MHC surface. Further, a neighbouring residue located on the same TCR loop (CDR3α E98) forms an energetically critical interaction with the MHC molecule. These data show how binding by a self-reactive TCR favors crossreactivity between self and microbial antigens.

Synthetic ligands able to interact with the P53 tetramerization domain. Towards understanding a protein surface recognition event

The applied interaction of synthetic molecules with defined regions of protein surfaces is an emerging strategy for the modulation of protein activity and/or stability. In spite of recent advances, the design of these molecules is not trivial. Among the most challenging aspects in designing these compounds is that they must compete with water molecules for interaction with polar patches of protein surfaces. Herein is reported the preparation of an arginine‐rich peptide that interacts in aqueous solution with a very hydrophilic patch at the surface of the tetramerization domain of the tumor suppressor protein p53. The interaction has been studied by several complementary techniques. By using this peptide as a template, a library of peptides has been prepared and evaluated in order to examine the different factors that contribute to the recognition event. The conclusions extracted from this work could be useful for the design of ligands directed at highly hydrophilic protein surface patches.

Gene activation of SMN by selective disruption of lncRNA-mediated recruitment of PRC2 for the treatment of spinal muscular atrophy

Significance Autosomal recessive mutations or deletions of the gene Survival Motor Neuron 1 (SMN1) cause spinal muscular atrophy, a neurodegenerative disorder. Transcriptional up-regulation of a nearly identical gene, SMN2, can functionally compensate for the loss of SMN1, resulting in increased SMN protein to ameliorate the disease severity. Here we demonstrate that the repressed state of SMN2 is reversible by interrupting the recruitment of a repressive epigenetic complex in disease-relevant cell types. Using chemically modified oligonucleotides to bind at a site of interaction on a long noncoding RNA that recruits the repressive complex, SMN2 is epigenetically altered to create a transcriptionally permissive state. Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.

Multicomponent Injectable Hydrogels for Antigen-Specific Tolerogenic Immune Modulation

Biomaterial scaffolds that enrich and modulate immune cells in situ can form the basis for potent immunotherapies to elicit immunity or reëstablish tolerance. Here, the authors explore the potential of an injectable, porous hydrogel to induce a regulatory T cell (Treg) response by delivering a peptide antigen to dendritic cells in a noninflammatory context. Two methods are described for delivering the BDC peptide from pore-forming alginate gels in the nonobese diabetic mouse model of type 1 diabetes: encapsulation in poly(lactide-co-glycolide) (PLG) microparticles, or direct conjugation to the alginate polymer. While particle-based delivery leads to antigen-specific T cells responses in vivo, PLG particles alter the phenotype of the cells infiltrating the gels. Following gel-based peptide delivery, transient expansion of endogenous antigen-specific T cells is observed in the draining lymph nodes. Antigen-specific T cells accumulate in the gels, and, strikingly, ≈60% of the antigen-specific CD4+ T cells in the gels are Tregs. Antigen-specific T cells are also enriched in the pancreatic islets, and administration of peptide-loaded gels does not accelerate diabetes. This work demonstrates that a noninflammatory biomaterial system can generate antigen-specific Tregs in vivo, which may enable the development of new therapies for the treatment of transplant rejection or autoimmune diseases.

Peptide data mining: from virtual design to knowledge extraction

Ignasi Belda, Ivan Traus, Susana Gordo, Teresa Tarrago, Sergio Madurga, Xavier Llora, and Ernest Giralt Institut de Recerca Biomedica, Parc Cientific de Barcelona, E 08028 Barcelona, Spain. b Conducive Corp. NY 10004, USA. c Illinois Genetic Algorithms Laboratory, Department of General Engineering, University of Illinois at Urbana-Champaign. IL 61801, USA. {ibelda,sgordo,ttarrago,smadurga,egiralt}@pcb.ub.edu, itraus@conducivecorp.com, xllora@illigal.ge.uiuc.edu