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Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease. Author summary Enteroviruses are associated with a variety of human diseases, including gastroenteritis, the common cold, hand-foot-and-mouth disease, acute hemorrhagic conjunctivitis, and skin rash. In some cases, the infection can lead to myocarditis, encephalitis, progressive muscle weakness, and paralysis. Exactly how enteroviruses invade human tissues, defeat the host immune system, and alter normal cell biology is unknown. Understanding these cellular and molecular mechanisms will blaze the trail for the development of novel vaccine and therapeutic strategies. Here, we have applied a global N-terminomics approach to investigate how various enteroviruses recruit their proteases to remodel an infected cell, disarm host immunity, and create a favorable environment for their replication. This effort identified several new protease substrates, which we then confirmed by other experimental approaches. To our knowledge, this is the first systematic analysis of host proteins targeted for cleavage during enterovirus infection. The data generated in this study will serve as a valuable resource for the research community in the quest to uncover the molecular details of enterovirus cell biology and disease pathogenesis.

Expression of the axon-guidance geneSlit2in endothelium, induced by endothelial sensing of tumour-derived double-stranded RNA, promotes metastatic dissemination in mouse models of breast and lung cancer. Blood vessels support tumours by providing nutrients and oxygen, while also acting as conduits for the dissemination of cancer(1). Here we use mouse models of breast and lung cancer to investigate whether endothelial cells also have active 'instructive' roles in the dissemination of cancer. We purified genetically tagged endothelial ribosomes and their associated transcripts from highly and poorly metastatic tumours. Deep sequencing revealed that metastatic tumours induced expression of the axon-guidance geneSlit2in endothelium, establishing differential expression between the endothelial (highSlit2expression) and tumoural (lowSlit2expression) compartments. Endothelial-derived SLIT2 protein and its receptor ROBO1 promoted the migration of cancer cells towards endothelial cells and intravasation. Deleting endothelialSlit2suppressed metastatic dissemination in mouse models of breast and lung cancer. Conversely, deletion of tumouralSlit2enhanced metastatic progression. We identified double-stranded RNA derived from tumour cells as an upstream signal that induces expression of endothelial SLIT2 by acting on the RNA-sensing receptor TLR3. Accordingly, a set of endogenous retroviral element RNAs were upregulated in metastatic cells and detected extracellularly. Thus, cancer cells co-opt innate RNA sensing to induce a chemotactic signalling pathway in endothelium that drives intravasation and metastasis. These findings reveal that endothelial cells have a direct instructive role in driving metastatic dissemination, and demonstrate that a single gene (Slit2) can promote or suppress cancer progression depending on its cellular source.

Bacterial pathogens have evolved to secrete strong anti-inflammatory proteins that target the immune system. It was long speculated whether these virulence factors could serve as therapeutics in diseases in which abnormal immune activation plays a role. We adopted the secreted chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) as a model virulence factor-based therapeutic agent for diseases in which C5AR1 stimulation plays an important role. We show that the administration of CHIPS in human C5AR1 knock-in mice successfully dampens C5a-mediated neutrophil migration during immune complex-initiated inflammation. Subsequent CHIPS toxicology studies in animal models were promising. However, during a small phase I trial, healthy human volunteers showed adverse effects directly after CHIPS administration. Subjects showed clinical signs of anaphylaxis with mild leukocytopenia and increased C-reactive protein concentrations, which are possibly related to the presence of relatively high circulating anti-CHIPS antibodies and suggest an inflammatory response. Even though our data in mice show CHIPS as a potential anti-inflammatory agent, safety issues in human subjects temper the use of CHIPS in its current form as a therapeutic candidate. The use of staphylococcal proteins, or other bacterial proteins, as therapeutics or immune-modulators in humans is severely hampered by pre-existing circulating antibodies.

A major obstacle to treating Alzheimer's disease (AD) is our lack of understanding of the molecular mechanisms underlying selective neuronal vulnerability, a key characteristic of the disease. Here, we present a framework integrating high-quality neuron-type-specific molecular profiles across the lifetime of the healthy mouse, which we generated using bacTRAP, with postmortem human functional genomics and quantitative genetics data. We demonstrate human-mouse conservation of cellular taxonomy at the molecular level for neurons vulnerable and resistant in AD, identify specific genes and pathways associated with AD neuropathology, and pinpoint a specific functional gene module underlying selective vulnerability, enriched in processes associated with axonal remodeling, and affected by amyloid accumulation and aging. We have made all cell-type-specific profiles and functional networks available at http://alz.princeton.edu. Overall, our study provides a molecular framework for understanding the complex interplay between Ab, aging, and neurodegeneration within the most vulnerable neurons in AD.

Interest in the C-terminal domain (CTD) of the RPB1 subunit of the RNA polymerase II (Pol II) has been revived in recent years, owing to its numerous posttranslational modifications and its "phase-separation" properties. A large number of studies have shown that the status of CTD modifications is associated with the activity of Pol II during the transcription cycle. However, because this domain is essential in living cells, the functional requirement of the full CTD for the control of Pol II activity at endogenous mammalian genes has never been addressed directly in living cells. Using an inducible Pol II-degradation system that we previously established, we investigated here the roles of the CTD in the post-initiation control of Pol II. The selective ablation of the RPB1 CTD, post-initiation, at promoter-proximal pause-sites revealed that this domain, and by extension the CTD heptads and their modifications, is functionally neither absolutely required to maintain pausing in the absence of CDK9 activity nor essential for the release of Pol II into productive elongation. (C) 2020 Elsevier Ltd. All rights reserved.

Interferon-stimulated gene 15 (ISG15)was the first ubiquitin-like modifier protein identified that acts by protein conjugation (ISGylation) and is thought to modulate IFN-induced inflammation. Here, we report a new patient from a non-consanguineous Argentinian family, who was followed for recurrent ulcerative skin lesions, cerebral calcifications and lung disease. Whole Exome Sequencing (WES) revealed two novel compound heterozygous variants (c.285del and c.299_312del, NM_005101.4 GRCh37(hg19), both classified as pathogenic according to ACMG criteria) in theISG15gene, resulting in a complete deficiency due to disruption of the second ubiquitin domain of the corresponding protein. The clinical phenotype of this patient is unique given the presence of recurrent pulmonary manifestations and the absence of mycobacterial infections, thus resulting in a phenotype distinct from that previously described in patients with biallelic loss-of-function (LOF)ISG15variants. This case highlights the role ofISG15as an immunomodulating factor whose LOF variants result in heterogeneous clinical presentations.

Allogeneic hematopoietic cell transplantation (alloHCT) benefits increasing numbers of patients with otherwise lethal diseases. Graft-versus-host disease (GVHD), however, remains one of the most potentially life-threatening complications due to its own comorbidities and the side effects of its treatment. In this issue of the JCI, two groups have turned dogma on its head by providing evidence for alternative mechanisms of acute GVHD (aGVHD) in humans. The principle of donor T cell reactivity elicited by host antigen-presenting cells (APCs) expressing MHC-encoded major HLA disparities or expressing minor histocompatibility antigen (miHA) differences presented by identical HLA molecules remains intact. These reports, however, demonstrate that GVHD can additionally result from peripheral host T cells resident in skin and gut being stimulated against donor APCs in the form of monocyte-derived macrophages. Moreover, these donor monocyte-derived macrophages can themselves mediate cytopathic effects against resident host T cells in skin explants and against a keratinocyte-derived cell line.

Ageing in humans is associated with an increase in circulating methylmalonic acid, which induces expression of SOX4 and promotes tumour progression. The risk of cancer and associated mortality increases substantially in humans from the age of 65 years onwards(1-6). Nonetheless, our understanding of the complex relationship between age and cancer is still in its infancy(2,3,7,8). For decades, this link has largely been attributed to increased exposure time to mutagens in older individuals. However, this view does not account for the established role of diet, exercise and small molecules that target the pace of metabolic ageing(9-12). Here we show that metabolic alterations that occur with age can produce a systemic environment that favours the progression and aggressiveness of tumours. Specifically, we show that methylmalonic acid (MMA), a by-product of propionate metabolism, is upregulated in the serum of older people and functions as a mediator of tumour progression. We traced this to the ability of MMA to induce SOX4 expression and consequently to elicit transcriptional reprogramming that can endow cancer cells with aggressive properties. Thus, the accumulation of MMA represents a link between ageing and cancer progression, suggesting that MMA is a promising therapeutic target for advanced carcinomas.

Mesoscale macromolecular complexes and organelles, tens to hundreds of nanometers in size, crowd the eukaryotic cytoplasm. It is therefore unclear how mesoscale particles remain sufficiently mobile to regulate dynamic processes such as cell division. Here, we study mobility across dividing cells that contain densely packed, dynamic microtubules, comprising the metaphase spindle. In dividing human cells, we tracked 40 nm genetically encoded multimeric nanoparticles (GEMs), whose sizes are commensurate with the inter-filament spacing in metaphase spindles. Unexpectedly, the effective diffusivity of GEMs was similar inside the dense metaphase spindle and the surrounding cytoplasm. Eliminating microtubules or perturbing their polymerization dynamics decreased diffusivity by similar to 30%, suggesting that microtubule polymerization enhances random displacements to amplify diffusive-like motion. Our results suggest that microtubules effectively fluidize the mitotic cytoplasm to equalize mesoscale mobility across a densely packed, dynamic, non-uniform environment, thus spatially maintaining a key biophysical parameter that impacts biochemistry, ranging from metabolism to the nucleation of cytoskeletal filaments.