In vivo interaction screening reveals liver-derived constraints to metastasis.

It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.

MHC class II proteins mediate cross-species entry of bat influenza viruses.

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.

High-throughput identification of RNA localization elements in neuronal cells.

Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse and human neuronal cells. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.

A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons.

Asymmetric subcellular mRNA localization allows spatial regulation of gene expression and functional compartmentalization. In neurons, localization of specific mRNAs to neurites is essential for cellular functioning. However, it is largely unknown how transcript sorting works in a sequence-specific manner. Here, we combined subcellular transcriptomics and massively parallel reporter assays and tested ∼50 000 sequences for their ability to localize to neurites. Mapping the localization potential of >300 genes revealed two ways neurite targeting can be achieved: focused localization motifs and broadly encoded localization potential. We characterized the interplay between RNA stability and localization and identified motifs able to bias localization towards neurite or soma as well as the trans-acting factors required for their action. Based on our data, we devised machine learning models that were able to predict the localization behavior of novel reporter sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native full-length transcripts.

Influenza A viruses balance ER stress with host protein synthesis shutoff.

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.

BRD9 is a druggable component of interferon-stimulated gene expression and antiviral activity.

Interferon (IFN) induction of IFN-stimulated genes (ISGs) creates a formidable protective antiviral state. However, loss of appropriate control mechanisms can result in constitutive pathogenic ISG upregulation. Here, we used genome-scale loss-of-function screening to establish genes critical for IFN-induced transcription, identifying all expected members of the JAK-STAT signaling pathway and a previously unappreciated epigenetic reader, bromodomain-containing protein 9 (BRD9), the defining subunit of non-canonical BAF (ncBAF) chromatin-remodeling complexes. Genetic knockout or small-molecule-mediated degradation of BRD9 limits IFN-induced expression of a subset of ISGs in multiple cell types and prevents IFN from exerting full antiviral activity against several RNA and DNA viruses, including influenza virus, human immunodeficiency virus (HIV1), and herpes simplex virus (HSV1). Mechanistically, BRD9 acts at the level of transcription, and its IFN-triggered proximal association with the ISG transcriptional activator, STAT2, suggests a functional localization at selected ISG promoters. Furthermore, BRD9 relies on its intact acetyl-binding bromodomain and unique ncBAF scaffolding interaction with GLTSCR1/1L to promote IFN action. Given its druggability, BRD9 is an attractive target for dampening ISG expression under certain autoinflammatory conditions.

Protein disulfide isomerase A6 controls the decay of IRE1α signaling via disulfide-dependent association.

The response to endoplasmic reticulum (ER) stress relies on activation of unfolded protein response (UPR) sensors, and the outcome of the UPR depends on the duration and strength of signal. Here, we demonstrate a mechanism that attenuates the activity of the UPR sensor inositol-requiring enzyme 1α (IRE1α). A resident ER protein disulfide isomerase, PDIA6, limits the duration of IRE1α activity by direct binding to cysteine 148 in the lumenal domain of the sensor, which is oxidized when IRE1 is activated. PDIA6-deficient cells hyperrespond to ER stress with sustained autophosphorylation of IRE1α and splicing of XBP1 mRNA, resulting in exaggerated upregulation of UPR target genes and increased apoptosis. In vivo, PDIA6-deficient C. elegans exhibits constitutive UPR and fails to complete larval development, a program that normally requires the UPR. Thus, PDIA6 activity provides a mechanism that limits UPR signaling and maintains it within a physiologically appropriate range.

PDIA6 regulates insulin secretion by selectively inhibiting the RIDD activity of IRE1.

Protein disulfide isomerase A6 (PDIA6) interacts with protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme (IRE)-1 and inhibits their unfolded protein response signaling. In this study, shRNA silencing of PDIA6 expression in insulin-producing mouse cells reduced insulin production (5-fold) and, consequently, glucose-stimulated insulin secretion (3-4-fold). This inhibition of insulin release was independent of the PDIA6-PERK interaction or PERK activity. Acute inhibition of PERK did not change the short-term response of ß cells to glucose. Rather, PDIA6 affected insulin secretion by modulating one of the activities of IRE1. At 11 mM glucose and lower, the regulated IRE1-dependent decay (RIDD) of the mRNA activity of IRE1 was activated, but not its X-box binding protein (XBP)-1 splicing activity. In the absence of PDIA6, RIDD activity toward insulin transcripts was enhanced up to 4-fold, as shown by molecular assays in cultured cells and the use of a fluorescent reporter in intact islets. Such physiologic activation of IRE1 by glucose contrasted with IRE1 activation by chemical stress, when both IRE1 activities were induced. Thus, whereas the stimulus determines the quality of IRE1 signaling, PDIA6 attenuates multiple enzymatic activities of IRE1, maintaining its signaling within a physiologically tolerable range.

Redox controls UPR to control redox.

In many physiological contexts, intracellular reduction-oxidation (redox) conditions and the unfolded protein response (UPR) are important for the control of cell life and death decisions. UPR is triggered by the disruption of endoplasmic reticulum (ER) homeostasis, also known as ER stress. Depending on the duration and severity of the disruption, this leads to cell adaptation or demise. In this Commentary, we review reductive and oxidative activation mechanisms of the UPR, which include direct interactions of dedicated protein disulfide isomerases with ER stress sensors, protein S-nitrosylation and ER Ca(2+) efflux that is promoted by reactive oxygen species. Furthermore, we discuss how cellular oxidant and antioxidant capacities are extensively remodeled downstream of UPR signals. Aside from activation of NADPH oxidases, mitogen-activated protein kinases and transcriptional antioxidant responses, such remodeling prominently relies on ER-mitochondrial crosstalk. Specific redox cues therefore operate both as triggers and effectors of ER stress, thus enabling amplification loops. We propose that redox-based amplification loops critically contribute to the switch from adaptive to fatal UPR.

Limitation of individual folding resources in the ER leads to outcomes distinct from the unfolded protein response.

ER stress leads to upregulation of multiple folding and quality control components, known as the unfolded protein response (UPR). Glucose Regulated Protein 78 (GRP78) (also known as binding immunoglobulin protein, BiP, and HSPA5) and GRP94 are often upregulated coordinately as part of this homeostatic response. Given that endoplasmic reticulum (ER) chaperones have distinct sets of clients, we asked how cells respond to ablation of individual chaperones. The cellular responses to silencing BiP, GRP94, HSP47, PDIA6 and OS-9, were distinct. When BiP was silenced, a widespread UPR was observed, but when GRP94 was either inhibited or depleted by RNA interference (RNAi), the expression of only some genes was induced, notably those encoding BiP and protein disulfide isomerase A6 (PDIA6). Silencing of HSP47 or OS-9 did not lead to any compensatory induction of other genes. The selective response to GRP94 depletion was distinct from a typical ER stress response, both because other UPR target genes were not affected and because the canonical UPR signaling branches were not activated. The response to silencing of GRP94 did not preclude further UPR induction when chemical stress was imposed. Importantly, re-expression of wild-type GRP94 in the silenced cells prevented the upregulation of BiP and PDIA6, whereas re-expression of an ATPase-deficient GRP94 mutant did not, indicating that cells monitor the activity state of GRP94. These findings suggest that cells are able to distinguish among folding resources and generate distinct responses.

GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum.

Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94 shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitated by the conditions in the endoplasmic reticulum. GRP94's mode of action varies from the general HSP90 theme in the conformational changes induced by nucleotide binding, and in its interactions with co-chaperones, which are very different from known cytosolic co-chaperones. GRP94 is more selective than many of the ER chaperones and the basis for this selectivity remains obscure. Recent development of molecular tools and functional assays has expanded the spectrum of clients that rely on GRP94 activity, but it is still not clear how the chaperone binds them, or what aspect of folding it impacts. These mechanistic questions and the regulation of GRP94 activity by other proteins and by post-translational modification differences pose new questions and present future research avenues. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

Deletion of muscle GRP94 impairs both muscle and body growth by inhibiting local IGF production.

Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were ∼30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.

GRP94 in ER quality control and stress responses.

A system of endoplasmic reticulum (ER) chaperones has evolved to optimize the output of properly folded secretory and membrane proteins. An important player in this network is Glucose Regulated Protein 94 (GRP94). Over the last decade, new structural and functional data have begun to delineate the unique characteristics of GRP94 and have solidified its importance in ER quality control pathways. This review describes our current understanding of GRP94 and the four ways in which it contributes to the ER quality control: (1) chaperoning the folding of proteins; (2) interacting with other components of the ER protein folding machinery; (3) storing calcium; and (4) assisting in the targeting of malfolded proteins to ER-associated degradation (ERAD).

Development of a Grp94 inhibitor.

Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/ß (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/ß client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.

Glucose regulated protein 94 is required for muscle differentiation through its control of the autocrine production of insulin-like growth factors.

The endoplasmic reticulum chaperone GRP94 is essential for early embryonic development and in particular affects differentiation of muscle lineages. To determine why an ubiquitously expressed protein has such a specific effect, we investigated the function of GRP94 in the differentiation of established myogenic cell lines in culture. Using both genetic suppression of expression, via RNA interference, and inhibition of function, via specific chemical inhibitors, we show that GRP94 expression and activity are needed for the in vitro fusion of myoblasts precursors into myotubes and the expression of contractile proteins that mark terminal differentiation. The inhibition can be complemented by addition of insulin-like growth factors to the cultures. GRP94 is not needed for the initial steps of myogenesis, only for the steps downstream of MyoD up-regulation, coinciding with the known need for synergistic input from growth factor signaling. Indeed, GRP94 is needed for the production of insulin-like growth factors I and II (IGF-I and IGF-II) by the differentiating cells. Moreover, the depletion of the chaperone does not increase the rate of apoptosis that always accompanies myogenic differentiation. Thus, the major effect of GRP94 on muscle differentiation is mediated by its regulation of IGF production.

CCL8/MCP-2 is a target for mir-146a in HIV-1-infected human microglial cells.

MicroRNA-mediated regulation of gene expression appears to be involved in a variety of cellular processes, including development, differentiation, proliferation, and apoptosis. Mir-146a is thought to be involved in the regulation of the innate immune response, and its expression is increased in tissues associated with chronic inflammation. Among the predicted gene targets for mir-146a, the chemokine CCL8/MCP-2 is a ligand for the CCR5 chemokine receptor and a potent inhibitor of CD4/CCR5-mediated HIV-1 entry and replication. In the present study, we have analyzed changes in the expression of mir-146a in primary human fetal microglial cells upon infection with HIV-1 and found increased expression of mir-146a. We further show that CCL8/MCP-2 is a target for mir-146a in HIV-1 infected microglia, as overexpression of mir-146a prevented HIV-induced secretion of MCP-2 chemokine. The clinical relevance of our findings was evaluated in HIV-encephalitis (HIVE) brain samples in which decreased levels of MCP-2 and increased levels of mir-146a were observed, suggesting a role for mir-146a in the maintenance of HIV-mediated chronic inflammation of the brain.

HIV-1 Tat C-terminus is cleaved by calpain 1: implication for Tat-mediated neurotoxicity.

HIV-Encephalopathy (HIVE) is a common neurological disorder associated with HIV-1 infection and AIDS. The activity of the HIV trans-activating protein Tat is thought to contribute to neuronal pathogenesis. While Tat proteins from primary virus isolates consist of 101 or more amino acids, 72 and 86 amino acids forms of Tat are commonly used for in vitro studies. Although Tat72 contains the minimal domain required for viral replication, other activities of Tat appear to vary according to its length, sub-cellular localization, cell type and the stage of cellular differentiation. In this study, we investigated the stability of intracellular Tat101 during proliferation and differentiation of neuronal cells in culture. We have utilized rat neuronal progenitors as a model of neuronal cell proliferation and differentiation, as well as rat primary cortical neurons as a model of fully differentiated cells. Our results indicate that, upon internalization, Tat101 was degraded more rapidly in proliferating cells than in cells which either underwent neuronal differentiation or were fully differentiated. Intracellular degradation of Tat was prevented by the calpain 1 inhibitor, ALLN, in both proliferating and differentiated cells. Inhibition of calpain 1 by calpastatin peptide also prevented Tat cleavage. In vitro calpain digestion and mass spectrometry analysis further demonstrated that the sequence of Tat sensitive to calpain cleavage was located in the C-terminus of this viral protein, between amino acids 68 and 69. Moreover, cleavage of Tat101 by calpain 1 increased neurotoxic effect of this viral protein and presence of the calpain inhibitor protected neuronal cells from Tat-mediated toxicity.

Profiling host ANP32A splicing landscapes to predict influenza A virus polymerase adaptation.

Species' differences in cellular factors limit avian influenza A virus (IAV) zoonoses and human pandemics. The IAV polymerase, vPol, harbors evolutionary sites to overcome restriction and determines virulence. Here, we establish host ANP32A as a critical driver of selection, and identify host-specific ANP32A splicing landscapes that predict viral evolution. We find that avian species differentially express three ANP32A isoforms diverging in a vPol-promoting insert. ANP32As with shorter inserts interact poorly with vPol, are compromised in supporting avian-like IAV replication, and drive selection of mammalian-adaptive vPol sequences with distinct kinetics. By integrating selection data with multi-species ANP32A splice variant profiling, we develop a mathematical model to predict avian species potentially driving (swallow, magpie) or maintaining (goose, swan) mammalian-adaptive vPol signatures. Supporting these predictions, surveillance data confirm enrichment of several mammalian-adaptive vPol substitutions in magpie IAVs. Profiling host ANP32A splicing could enhance surveillance and eradication efforts against IAVs with pandemic potential.

Cooperation of docosahexaenoic acid and vitamin E in the regulation of UDP-glucuronosyltransferase mRNA expression.

Docosahexaenoic acid (DHA) is a well known chemopreventive nutrient within diet formulations, but it may also exert toxic effects on cultured cells, while this is limited when also another relevant nutrient as vitamin E is present. This effect, beside the involvement of the two nutrients in oxidative processes, likely affects the expression of specific genes. To obtain information on combined activities of DHA and vitamin E on some gene products previously resulted to be in vivo regulated from dietary unsaturated fats, the effect of the two nutrients was evaluated in human cell line HepG2. Independently, DHA and vitamin E resulted to affect only slightly UDP-glucuronosyltransferase 1A1 (UGT1A1) mRNA expression. Nevertheless, their combination produced a considerable reduction of this mRNA. DHA also downregulated stearoyl-CoA desaturase (SCD) and sterol regulatory element binding protein (SREBP-1) expression, while vitamin E did not affect these products. However, their combination abolished the downregulation of SCD but did not affect that of SREBP-1. Therefore the effect of the two nutrients is related to specific gene regulation processes resulting in a cooperation which might be related to their physiological effects as dietary components.