Structural determinants of peptide-dependent TAP1-TAP2 transit passage targeted by viral proteins and altered by cancer-associated mutations

The TAP1-TAP2 complex transports antigenic peptide substrates into the endoplasmic reticulum (ER). In ER, the peptides are further processed and loaded on the major histocompatibility class (MHC) I molecules by the peptide loading complex (PLC). The TAP transporters are linked with the PLC; a target for cancers and viral immune evasion. But the mechanisms whereby the cancer-derived mutations in TAP1-TAP2 or viral factors targeting the PLC, interfere peptide transport are only emerging. This study describes that transit of peptides through TAP can take place via two different channels (4 or 8 helices) depending on peptide length and sequence. Molecular dynamics and binding affinity predictions of peptide-transporters demonstrated that smaller peptides (8–10 mers; e.g. AAGIGILTV, SIINFEKL) can transport quickly through the transport tunnel compared to longer peptides (15-mer; e.g. ENPVVHFFKNIVTPR). In line with a regulated and selective peptide transport by TAPs, the immunopeptidome upon IFN-γ treatment in melanoma cells induced the shorter length (9-mer) peptide presentation over MHC-I that exhibit a relatively weak binding affinity with TAP. A conserved distance between N and C terminus residues of the studied peptides in the transport tunnel were reported. Furthermore, by adversely interacting with the TAP transport passage or affecting TAPNBD domains tilt movement, the viral proteins and cancer-derived mutations in TAP1-TAP2 may induce allosteric effects in TAP that block conformation of the tunnel (closed towards ER lumen). Interestingly, some cancer-associated mutations (e.g. TAP1R372Q and TAP2R373H) can specifically interfere with selective transport channels (i.e. for longer-peptides). These results provide a model for how viruses and cancer-associated mutations targeting TAP interfaces can affect MHC-I antigen presentation, and how the IFN-γ pathway alters MHC-I antigen presentation via the kinetics of peptide transport

In silico-in vitro screening of protein-protein interactions: towards the next generation of therapeutics.

International audienceProtein-protein interactions (PPIs) have a pivotal role in many biological processes suggesting that targeting macromolecular complexes will open new avenues for the design of the next generation of therapeutics. A wide range of "in silico methods" can be used to facilitate the design of protein-protein modulators. Among these methods, virtual ligand screening, protein-protein docking, structural predictions and druggable pocket predictions have become established techniques for hit discovery and optimization. In this review, we first summarize some key data about protein-protein interfaces and introduce some recently reported computer methods pertaining to the field. URLs for several recent free packages or servers are also provided. Then, we discuss four studies aiming at developing PPI modulators through the combination of in silico and in vitro screening experiments

Interplay of inertia and deformability on rheological properties of a suspension of capsules

The interplay of inertia and deformability has a substantial impact on the transport of soft particles suspended in a fluid. However, to date a thorough understanding of these systems is still missing and only a limited number of experimental and theoretical studies is available. We combine the finite-element, immersed-boundary and lattice-Boltzmann methods to simulate three-dimensional suspensions of soft particles subjected to planar Poiseuille flow at finite Reynolds numbers. Our findings confirm that the particle deformation and inclination increase when inertia is present. We observe that the Segr\'e-Silberberg effect is suppressed with respect to the particle deformability. Depending on the deformability and strength of inertial effects, inward or outward lateral migration of the particles takes place. In particular, for increasing Reynolds numbers and strongly deformable particles, a hitherto unreported distinct flow focusing effect emerges which is accompanied by a non-monotonic behaviour of the apparent suspension viscosity and thickness of the particle-free layer close to the channel walls. This effect can be explained by the behaviour of a single particle and the change of the particle collision mechanism when both deformability and inertia effects are relevant.Comment: 20 pages, 9 figure

A single synonymous mutation determines the phosphorylation and stability of the nascent protein

p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein

Highly Conserved Homotrimer Cavity Formed by the SARS-CoV-2 Spike Glycoprotein: A Novel Binding Site

An important stage in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) life cycle is the binding of the spike (S) protein to the angiotensin converting enzyme-2 (ACE2) host cell receptor. Therefore, to explore conserved features in spike protein dynamics and to identify potentially novel regions for drugging, we measured spike protein variability derived from 791 viral genomes and studied its properties by molecular dynamics (MD) simulation. The findings indicated that S2 subunit (heptad-repeat 1 (HR1), central helix (CH), and connector domain (CD) domains) showed low variability, low fluctuations in MD, and displayed a trimer cavity. By contrast, the receptor binding domain (RBD) domain, which is typically targeted in drug discovery programs, exhibits more sequence variability and flexibility. Interpretations from MD simulations suggest that the monomer form of spike protein is in constant motion showing transitions between an “up” and “down” state. In addition, the trimer cavity may function as a “bouncing spring” that may facilitate the homotrimer spike protein interactions with the ACE2 receptor. The feasibility of the trimer cavity as a potential drug target was examined by structure based virtual screening. Several hits were identified that have already been validated or suggested to inhibit the SARS-CoV-2 virus in published cell models. In particular, the data suggest an action mechanism for molecules including Chitosan and macrolides such as the mTOR (mammalian target of Rapamycin) pathway inhibitor Rapamycin. These findings identify a novel small molecule binding-site formed by the spike protein oligomer, that might assist in future drug discovery programs aimed at targeting the coronavirus (CoV) family of viruses

Expression of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) gene is associated with developmental change in the life cycle of the model legume Medicago truncatula

SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes have been demonstrated to play a role in somatic embryogenesis in several plant species. As more is learnt about these genes, the view of their role in plant development has broadened. The Medicago truncatula MtSERK1 gene has been associated with somatic embryogenesis and in vitro root formation. In order to study the role of MtSERK1 in development further, the MtSERK1 promoter sequence has been isolated and cloned into a promoter–GUS analysis vector. SERK1 promoter-driven GUS expression was studied in A. tumefaciens-transformed cultures and regenerated plants, in A. rhizogenes-transformed root clones, and in nodulation. In embryogenic cultures, GUS staining is detected after 2 d of culture at the edge of the explant and around vascular tissue. Expression at the explant edge intensifies over subsequent days and then is lost from the edge as callus formation moves inward. MtSERK1 expression appears to be associated with new callus formation. When somatic embryos form, GUS staining occurs throughout embryo development. Zygotic embryos show expression until the heart stage. The in planta studies reveal a number of interesting expression patterns. There appear to be three types. (i) Expression associated with the primary meristems of the root and shoot and the newly formed meristems of the lateral roots and nodule. (ii) Expression at the junction between one type of tissue or organ and another. (iii) Expression associated with the vascular tissue procambial cells. The data led us to conclude that MtSERK1 expression is associated with developmental change, possibly reflecting cellular reprogramming

A bovine lymphosarcoma cell line infected with theileria annulata exhibits an irreversible reconfiguration of host cell gene expression

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner

Identification of Ventricular Tachycardia Using Intracavitary Ventricular Electrograms: Analysis of Time and Frequency Domain Patterns

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74961/1/j.1540-8159.1988.tb06279.x.pd