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LRRK2 Is Recruited to Phagosomes and Co-recruits RAB8 and RAB10 in Human Pluripotent Stem Cell-Derived Macrophages.
The Parkinson's disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease and is expressed in immune subsets. Here, we characterize a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes using human induced pluripotent stem cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-γ in these cells, including a 187-kDa cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles but is recruited to LAMP1+/RAB9+ "maturing" phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for RAB8a and RAB10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes.
Phosphoproteomics of CD2 signaling reveals AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells.
Understanding the costimulatory signaling that enhances the activity of cytotoxic T cells (CTLs) could identify potential targets for immunotherapy. Here, we report that CD2 costimulation plays a critical role in target cell killing by freshly isolated human CD8+ T cells, which represent a challenging but valuable model to gain insight into CTL biology. We found that CD2 stimulation critically enhanced signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules toward the microtubule-organizing center (MTOC). To gain insight into the underlying 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, cytoskeletal organization, autophagy, and metabolism. Signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) was a critical node in the CD2 network, which promoted granule polarization toward the MTOC in CD8+ T cells. Granule trafficking was driven by active AMPK enriched on adjacent lysosomes, revealing previously uncharacterized signaling cross-talk between vesicular compartments in CD8+ T cells. Our results thus establish CD2 signaling as key for mediating cytotoxic killing and granule polarization in freshly isolated CD8+ T cells and strengthen the rationale to choose CD2 and AMPK as therapeutic targets to enhance CTL activity.
<jats:p>Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes from CTLs to target cells, termed supramolecular attack particles (SMAPs). SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis and CRISPR editing identified a C-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nm diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo based on specificity of shell components.</jats:p>
© 2019, Springer Nature Switzerland AG. Histone demethylases (KDMs) catalyse the removal of N-methyl marks on histones and play important roles in epigenetic regulation. Abnormal histone methylation and dysregulation of KDMs have been linked to multiple diseases, and KDMs are emerging as promising therapeutic targets. This chapter provides an overview of JmjC-domain-containing KDMs (JmjC-KDMs), with a particular focus on recent advances in JmjC-KDM inhibitor development from a structural perspective.
Autophagy is an evolutionarily conserved mechanism for intracellular substance degradation, responsible for the recycling of metabolic substances and the maintenance of intracellular stability. It has early been demonstrated to play a significant role in tumorigenesis, but whether it acts as a promoter or a suppressor during tumorigenesis seems to be context-specific. Moreover, autophagy is also implicated in promoting chemoresistance of cancer cells so as to attenuate therapeutic efficacy of chemotherapy. On the contrary, other reports highlight a tumor-killing role of autophagy during cancer treatment. Herein, this review aims to revisit the key features of autophagy, summarize the seemingly contradictory roles of autophagy during both tumorigenesis and cancer chemotherapy, and evaluate the feasibility of altering the level of cellular autophagy as part of cancer adjuvant treatment.
MiRNAs are a new class of small RNA molecules that regulate gene expression at the post-transcriptional and translational levels. MiRNAs have been implicated in the control of many vital biological processes including development, cell proliferation, differentiation, and apoptosis. A growing number of studies have shown that miRNAs also play an important role in carcinogenesis and other diseases. Among the miRNAs identified, miRNA-21 is dramatically up-regulated in cancer cells of various origins. It regulates a wide range of genes and pathways involved in cancer initiation, transformation, invasion, and metastasis. MiRNA-21 also acts as a pro-survival factor in cardiovascular diseases. Aberrant expression in these diseases makes miRNA-21 a potential marker for disease diagnosis and prognosis. This review highlights the complex roles that miRNA-21 plays in cancer and cardiovascular diseases and its potential clinical applications.
Neuraminidase of Influenza A Virus Binds Lysosome-Associated Membrane Proteins Directly and Induces Lysosome Rupture
<jats:title>ABSTRACT</jats:title><jats:p>As a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as “lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol.” Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death.</jats:p><jats:p><jats:bold>IMPORTANCE</jats:bold>The integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian influenza virus infections at early stage.</jats:p>
Features of 16,749 hospitalised UK patients with COVID-19 using the ISARIC WHO Clinical Characterisation Protocol
<jats:p>Objective: To characterize the clinical features of patients with severe COVID-19 in the UK. Design: Prospective observational cohort study with rapid data gathering and near real-time analysis, using a pre-approved questionnaire adopted by the WHO. Setting: 166 UK hospitals between 6th February and 18th April 2020. Participants: 16,749 people with COVID-19. Interventions: No interventions were performed, but with consent samples were taken for research purposes. Many participants were co-enrolled in other interventional studies and clinical trials. Results: The median age was 72 years [IQR 57, 82; range 0, 104], the median duration of symptoms before admission was 4 days [IQR 1,8] and the median duration of hospital stay was 7 days [IQR 4,12]. The commonest comorbidities were chronic cardiac disease (29%), uncomplicated diabetes (19%), non-asthmatic chronic pulmonary disease (19%) and asthma (14%); 47% had no documented reported comorbidity. Increased age and comorbidities including obesity were associated with a higher probability of mortality. Distinct clusters of symptoms were found: 1. respiratory (cough, sputum, sore throat, runny nose, ear pain, wheeze, and chest pain); 2. systemic (myalgia, joint pain and fatigue); 3. enteric (abdominal pain, vomiting and diarrhoea). Overall, 49% of patients were discharged alive, 33% have died and 17% continued to receive care at date of reporting. 17% required admission to High Dependency or Intensive Care Units; of these, 31% were discharged alive, 45% died and 24% continued to receive care at the reporting date. Of those receiving mechanical ventilation, 20% were discharged alive, 53% died and 27% remained in hospital. Conclusions: We present the largest detailed description of COVID-19 in Europe, demonstrating the importance of pandemic preparedness and the need to maintain readiness to launch research studies in response to outbreaks. Trial documentation: Available at https://isaric4c.net/protocols . Ethical approval in England and Wales (13/SC/0149), and Scotland (20/SS/0028). ISRCTN (pending).</jats:p>