How lamina-associated polypeptide 1 (LAP1) activates Torsin

Lamina-associated polypeptide 1 (LAP1) resides at the nuclear envelope and interacts with Torsins, poorly understood endoplasmic reticulum (ER)-localized AAA+ ATPases, through a conserved, perinuclear domain. We determined the crystal structure of the perinuclear domain of human LAP1. LAP1 possesses an atypical AAA+ fold. While LAP1 lacks canonical nucleotide binding motifs, its strictly conserved arginine 563 is positioned exactly where the arginine finger of canonical AAA+ ATPases is found. Based on modeling and electron microscopic analysis, we propose that LAP1 targets Torsin to the nuclear envelope by forming an alternating, heterohexameric (LAP1-Torsin)[subscript 3] ring, in which LAP1 acts as the Torsin activator. The experimental data show that mutation of arginine 563 in LAP1 reduces its ability to stimulate TorsinA ATPase hydrolysis. This knowledge may help scientists understand the etiology of DYT1 primary dystonia, a movement disorder caused by a single glutamate deletion in TorsinA.National Institute of General Medical Sciences (U.S.) (Award GM103403)United States. Dept. of Energy. Office of Basic Energy Sciences (Contract DE-AC02-06CH11357

Targeting small molecule drugs to T cells with antibody-directed cell-penetrating gold nanoparticles

We sought to develop a nanoparticle vehicle that could efficiently deliver small molecule drugs to target lymphocyte populations. The synthesized amphiphilic organic ligand-protected gold nanoparticles (amph-NPs) were capable of sequestering large payloads of small molecule drugs within hydrophobic pockets of their ligand shells. These particles exhibit membrane-penetrating activity in mammalian cells, and thus enhanced uptake of a small molecule TGF-β inhibitor in T cells in cell culture. By conjugating amph-NPs with targeting antibodies or camelid-derived nanobodies, the particles' cell-penetrating properties could be temporarily suppressed, allowing targeted uptake in specific lymphocyte subpopulations. Degradation of the protein targeting moieties following particle endocytosis allowed the NPs to recover their cell-penetrating activity in situ to enter the cytoplasm of T cells. In vivo, targeted amph-NPs showed 40-fold enhanced uptake in CD8+ T cells relative to untargeted particles, and delivery of TGF-β inhibitor-loaded particles to T cells enhanced their cytokine polyfunctionality in a cancer vaccine model. Thus, this system provides a facile approach to concentrate small molecule compounds in target lymphocyte populations of interest for immunotherapy in cancer and other diseases.Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001)Melanoma Research AllianceNational Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. (Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Grant CA174795)National Institutes of Health (U.S.) (Grant CA172164)Horizon 2020 Framework Programme (European Commission). FutureNanoNeeds Projec

Investigation of the Proteolytic Functions of an Expanded Cercarial Elastase Gene Family in Schistosoma mansoni

Schistosome parasites are a major cause of disease in the developing world. The larval stage of the parasite transitions between an intermediate snail host and a definitive human host in a dramatic fashion, burrowing out of the snail and subsequently penetrating human skin. This process is facilitated by secreted proteases. In Schistosoma mansoni, cercarial elastase is the predominant secreted protease and essential for host skin invasion. Genomic analysis reveals a greatly expanded cercarial elastase gene family in S. mansoni. Despite sequence divergence, SmCE isoforms show similar expression profiles throughout the S. mansoni life cycle and have largely similar substrate specificities, suggesting that the majority of protease isoforms are functionally redundant and therefore their expansion is an example of gene dosage. However, activity-based profiling also indicates that a subset of SmCE isoforms are activated prior to the parasite's exit from its intermediate snail host, suggesting that the protease may also have a role in this process

Proteomic profiling of high risk medulloblastoma reveals functional biology

Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

Transpeptidation catalyzed by ​sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of ​sortase A on a solid support (Sepharose beads). Immobilization of ​sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca²⁺-independent variant of ​sortase A with increased catalytic activity. This heptamutant variant of ​sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized ​sortase A takes 1–2 d. Batch reactions take 3–12 h and flow reactions proceed at 0.5 ml h⁻¹, depending on the geometry of the reactor used.United States. National Institutes of Health (RO1 AI087879

Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8 T cells

Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non-small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno-positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89 Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8 + T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89 Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies.National Institutes of Health (U.S.) (Grant R01-AI087879-06)National Institutes of Health (U.S.) (Grant DP1-GM106409-03)National Institutes of Health (U.S.) (Grant R01-GM100518-04)National Institutes of Health (U.S.) (Grant P01 CA080111

Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus

Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection. However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response.National Institutes of Health (U.S.

Resilient Pedagogy: Practical Teaching Strategies to Overcome Distance, Disruption, and Distraction

Resilient Pedagogy offers a comprehensive collection on the topics and issues surrounding resilient pedagogy framed in the context of the COVID-19 pandemic and the social justice movements that have swept the globe. As a collection, Resilient Pedagogy is a multi-disciplinary and multi-perspective response to actions taken in different classrooms, across different institution types, and from individuals in different institutional roles with the purpose of allowing readers to explore the topics to improve their own teaching practice and support their own students through distance, disruption, and distraction