Immune Checkpoint LAG3 and Its Ligand FGL1 in Cancer

LAG3 is the most promising immune checkpoint next to PD-1 and CTLA-4. High LAG3 and FGL1 expression boosts tumor growth by inhibiting the immune microenvironment. This review comprises four sections presenting the structure/expression, interaction, biological effects, and clinical application of LAG3/FGL1. D1 and D2 of LAG3 and FD of FGL1 are the LAG3-FGL1 interaction domains. LAG3 accumulates on the surface of lymphocytes in various tumors, but is also found in the cytoplasm in non-small cell lung cancer (NSCLC) cells. FGL1 is found in the cytoplasm in NSCLC cells and on the surface of breast cancer cells. The LAG3-FGL1 interaction mechanism remains unclear, and the intracellular signals require elucidation. LAG3/FGL1 activity is associated with immune cell infiltration, proliferation, and secretion. Cytokine production is enhanced when LAG3/FGL1 are co-expressed with PD-1. IMP321 and relatlimab are promising monoclonal antibodies targeting LAG3 in melanoma. The clinical use of anti-FGL1 antibodies has not been reported. Finally, high FGL1 and LAG3 expression induces EGFR-TKI and gefitinib resistance, and anti-PD-1 therapy resistance, respectively. We present a comprehensive overview of the role of LAG3/FGL1 in cancer, suggesting novel anti-tumor therapy strategies.

Energetic determinants of the Par-3 interaction with the Par complex

The animal cell polarity regulator Par-3 recruits the Par complex (Par-6 and atypical Protein Kinase C–aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it has been unclear how each contributes to the overall interaction. Using purified, intact Par complex and a quantitative binding assay, we found that energy for this interaction is provided by Par-3’s second and third PDZ protein interaction domains. Both Par-3 PDZ domains bind to aPKC’s PDZ Binding Motif (PBM) in the Par complex, with binding energy contributed from aPKC’s adjacent catalytic domain. In addition to highlighting the role of Par-3 PDZ interactions with the aPKC kinase domain and PBM in stabilizing Par-3 – Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization.

Heterologous Expression and Assembly of Human TLR Signaling Components in Saccharomyces cerevisiae

Toll-like receptor (TLR) signaling is key to detect pathogens and initiating inflammation. Ligand recognition triggers the assembly of supramolecular organizing centers (SMOCs) consisting of large complexes composed of multiple subunits. Building such signaling hubs relies on Toll Interleukin-1 Receptor (TIR) and Death Domain (DD) protein-protein interaction domains. We have expressed TIR domain-containing components of the human myddosome (TIRAP and MyD88) and triffosome (TRAM and TRIF) SMOCs in Saccharomyces cerevisiae, as a platform for their study. Interactions between the TLR4 TIR domain, TIRAP, and MyD88 were recapitulated in yeast. Human TIRAP decorated the yeast plasma membrane (PM), except for the bud neck, whereas MyD88 was found at cytoplasmic spots, which were consistent with endoplasmic reticulum (ER)-mitochondria junctions, as evidenced by co-localization with Mmm1 and Mdm34, components of the ER and Mitochondria Encounter Structures (ERMES). The formation of MyD88-TIRAP foci at the yeast PM was reinforced by co-expression of a membrane-bound TLR4 TIR domain. Mutations in essential residues of their TIR domains aborted MyD88 recruitment by TIRAP, but their respective subcellular localizations were unaltered. TRAM and TRIF, however, did not co-localize in yeast. TRAM assembled long PM-bound filaments that were disrupted by co-expression of the TLR4 TIR domain. Our results evidence that the yeast model can be exploited to study the interactions and subcellular localization of human SMOC components in vivo.

Two paths towards the future of remote studies using social VR

Over the last decade, remote experiments have become a widely used and integral method for many human-computer interaction domains. Nonetheless, extended reality (XR) researchers have been slow to adopt remote research methods. This can largely be attributed to standard remote experimentation techniques being ill-suited for the unique XR domain constraints. Existing research, albeit limited, has aimed to overcome these constraints and demonstrate the viability of traditional remote research methods for XR studies, yet most XR experiments have remained in-lab. This gap in XR methodology has never been more evident or detrimental than during the ongoing global COVID-19 pandemic. During the pandemic, safe and ethical co-present in-lab experimentation has become increasingly difficult, if not impossible. Many researchers struggled to transition to remote research methods resulting in delayed, canceled, or unsatisfactory experiments. Beyond this current crisis, remote research methods present several advantages, such as obtaining a larger sample and accessing specific user populations that have not been leveraged in XR research — leading to missed opportunities and potentially less rigorous results.Our previous research demonstrated the efficacy of using existing social virtual reality (VR) platforms to implement and conduct remote VR experiments. Social VR platforms provide an experienced and VR-equipped user base to recruit from and customizable distributed synchronous virtual environments to implement experiments, which makes them a natural fit for VR experiments. They allow researchers to be co-present in the same virtual environment as participants to proctor experiments, similar to how they would during a co-present in-lab study. However, existing social VR platforms were not built with this use-case in mind, resulting in several limitations, such as the inability to easily save data externally. These limitations prevent existing social VR platforms from being a viable long-term XR research method. Our previous work identified two potential paths towards establishing long-term social VR remote research methods. The first potential path is to partner with existing social VR platforms to create official channels for remote studies. The second potential path is to build a bespoke social VR platform specifically for conducting XR remote experiments. We believe both of these paths have their respective strengths and weakness and are viable long-term solutions for remote XR studies. In this position paper, we aim to contribute a detailed discussion of both of these paths, their benefits, limitations, and potential architecture. In so doing, we hope to provide the XR community our insights into how social VR research methods can be expanded and inspiration for the potential future of remote XR research.

Characterization of the interaction domains between the phosphoprotein and the nucleoprotein of human Metapneumovirus

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N complex (N Nuc ), which serves as template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to N Nuc and L, allowing the attachment of the L polymerase to the N Nuc template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between HMPV P and N Nuc , which has not been demonstrated yet. Here, we finely characterized the binding P- N Nuc interaction domains by using recombinant proteins, combined with a functional assay for the polymerase complex activity, and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to N Nuc , that P binds to the N-terminal domain of N (N NTD ), and identified conserved N residues critical for the interaction. Our results allowed to propose a structural model for the HMPV P-N Nuc interaction. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Nowadays, no vaccine or efficient antiviral treatments are available for this pneumovirus. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction, and identified a pocket on the surface of the N protein, a potential target of choice for the design of compounds interfering with N-P complexes and inhibiting viral replication.

Global mapping of the energetic and allosteric landscapes of protein binding domains

Allosteric communication between distant sites in proteins is central to nearly all biological regulation but still poorly characterised for most proteins, limiting conceptual understanding, biological engineering and allosteric drug development. Typically only a few allosteric sites are known in model proteins, but theoretical, evolutionary and some experimental studies suggest they may be much more widely distributed. An important reason why allostery remains poorly characterised is the lack of methods to systematically quantify long-range communication in diverse proteins. Here we address this shortcoming by developing a method that uses deep mutational scanning to comprehensively map the allosteric landscapes of protein interaction domains. The key concept of the approach is the use of 'multidimensional mutagenesis': mutational effects are quantified for multiple molecular phenotypes - here binding and protein abundance -and in multiple genetic backgrounds. This is an efficient experimental design that allows the underlying causal biophysical effects of mutations to be accurately inferred en masse by fitting thermodynamic models using neural networks. We apply the approach to two of the most common human protein interaction domains, an SH3 domain and a PDZ domain, to produce the first global atlases of allosteric mutations for any proteins. Allosteric mutations are widely dispersed with extensive long-range tuning of binding affinity and a large mutational target space of network-altering 'edgetic' variants. Mutations are more likely to be allosteric closer to binding interfaces, at Glycines in secondary structure elements and at particular sites including a chain of residues connecting to an opposite surface in the PDZ domain. This general approach of quantifying mutational effects for multiple molecular phenotypes and in multiple genetic backgrounds should allow the energetic and allosteric landscapes of many proteins to be rapidly and comprehensively mapped.

Compatibility of the Ligand Binding Sites in the Spike Glycoprotein of COVID-19 with those in the Aminopeptidase and the Caveolins 1, 2 Proteins

A novel severe viral pneumonia emerged in Wuhan city, China, in December 2019. The spike glycoprotein of the SARS-CoV-2 plays a crucial role in the viral entry to the host cell and eliciting a strong response for antibody-mediated neutralization in mice. Caveolins 1,2 are scaffolding proteins dovetailed as a co-stimulatory signal essential for T-cell receptor and activation. Aminopeptidase is a membrane protein acting as a receptor for human coronavirus within the S1 subunit of the spike glycoprotein. Vaccines for COVID-19 have become a priority for predisposition against the outbreak, so that our study aimed to find interaction sites between SP of SARS-CoV-2 and CAV1, CAV2, and AMPN. Methods: Amino acids motif search was employed to predict the possible CAV1, CAV2, and AMPN related interaction domains in the SARS-CoV-2 SP In silico analysis. Results: Interactions between proteins revealed 5 and16 residues. ZN ligand binding site is matched between AMPN and SARS- CoV-2 SP. HLA-A*74:01 allele is the best CTL epitope for SP. We identified seven B-cell epitopes specifically for SARS-CoV-2 SP. Conclusions: SARS-CoV-2 SP binding sites might be compatible with AMPN ligand binding sites. The limit score was detected for ligand binding sites of CAV1 and CAV2. Our findings might be critical for the further substantial study of vaccine production strategy.

AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP γ8 interactions

AMPA receptor (AMPAR) abundance and positioning at excitatory synapses regulates the strength of transmission. Changes in AMPAR localisation can enact synaptic plasticity, allowing long-term information storage, and is therefore tightly controlled. Multiple mechanisms regulating AMPAR synaptic anchoring have been described, but with limited coherence or comparison between reports, our understanding of this process is unclear. Here, combining synaptic recordings from mouse hippocampal slices and super-resolution imaging in dissociated cultures, we compare the contributions of three AMPAR interaction domains controlling transmission at hippocampal CA1 synapses. We show that the AMPAR C-termini play only a modulatory role, whereas the extracellular N-terminal domain (NTD) and PDZ interactions of the auxiliary subunit TARP γ8 are both crucial, and each is sufficient to maintain transmission. Our data support a model in which γ8 accumulates AMPARs at the postsynaptic density, where the NTD further tunes their positioning. This interplay between cytosolic (TARP γ8) and synaptic cleft (NTD) interactions provides versatility to regulate synaptic transmission and plasticity.

The stabilized Pol31–Pol3 interface counteracts Pol32 ablation with differential effects on repair

DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of pol31 is lethal, and compromising the Pol3–Pol31 interaction domains confers hypersensitivity to cold, hydroxyurea (HU), and methyl methanesulfonate, phenocopying pol32Δ. We have identified alanine-substitutions in pol31 that suppress these deficiencies in pol32Δ cells. We characterize two mutants, pol31-T415A and pol31-W417A, which map to a solvent-exposed loop that mediates Pol31–Pol3 and Pol31–Rev3 interactions. The pol31-T415A substitution compromises binding to the Pol3 CysB domain, whereas Pol31-W417A improves it. Importantly, loss of Pol32, such as pol31-T415A, leads to reduced Pol3 and Pol31 protein levels, which are restored by pol31-W417A. The mutations have differential effects on recovery from acute HU, break-induced replication and trans-lesion synthesis repair pathways. Unlike trans-lesion synthesis and growth on HU, the loss of break-induced replication in pol32Δ cells is not restored by pol31-W417A, highlighting pathway-specific roles for Pol32 in fork-related repair. Intriguingly, CHIP analyses of replication forks on HU showed that pol32Δ and pol31-T415A indirectly destabilize DNA pol α and pol ε at stalled forks.