Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

Newly synthesized proteins in the endoplasmic reticulum (ER) are sorted by coat protein complex II (COPII) at the ER exit site en route to the Golgi. Under cellular stresses, COPII proteins become targets of regulation to control the transport. Here, we show that the COPII outer coat proteins Sec31 and Sec13 are selectively sequestered into the biomolecular condensate of SCOTIN/SHISA-5, which interferes with COPII vesicle formation and inhibits ER-to-Golgi transport. SCOTIN is an ER transmembrane protein with a cytosolic intrinsically disordered region (IDR), which is required and essential for the formation of condensates. Upon IFN-γ stimulation, which is a cellular condition that induces SCOTIN expression and condensation, ER-to-Golgi transport was inhibited in a SCOTIN-dependent manner. Furthermore, cancer-associated mutations of SCOTIN perturb its ability to form condensates and control transport. Together, we propose that SCOTIN impedes the ER-to-Golgi transport through its ability to form biomolecular condensates at the ER membrane.

Homotypic SCOTIN assemblies form ER-endosome membrane contacts and regulate endosome dynamics.

The ER regulates the spatiotemporal organization of endolysosomal systems by membrane contact. In addition to tethering via heterotypic interactions on both organelles, we present a novel ER-endosome tethering mechanism mediated by homotypic interactions. The single-pass transmembrane protein SCOTIN is detected in the membrane of the ER and endosomes. In SCOTIN-knockout (KO) cells, the ER-late endosome contacts are reduced, and the perinuclear positioning of endosomes is disturbed. The cytosolic proline-rich domain (PRD) of SCOTIN forms homotypic assemblies in vitro and is necessary for ER-endosome membrane tethering in cells. A region of 28 amino acids spanning 150-177 within the SCOTIN PRD is essential to elicit membrane tethering and endosomal dynamics, as verified by reconstitution in SCOTIN-KO cells. The assembly of SCOTIN (PRD) is sufficient to mediate membrane tethering, as purified SCOTIN (PRD), but not SCOTIN (PRDΔ150-177), brings two different liposomes closer in vitro. Using organelle-specific targeting of a chimeric PRD domain shows that only the presence on both organellar membranes enables the ER-endosome membrane contact, indicating that the assembly of SCOTIN on heterologous membranes mediates organelle tethering.

The efficacy of detecting arrhythmia is higher with 7-day continuous electrocardiographic patch monitoring than with 24-h Holter monitoring.

Background: Detecting high-risk arrhythmia is important in diagnosing patients with palpitations. We compared the diagnostic accuracies of 7-day patch-type electrocardiographic (ECG) monitoring and 24-h Holter monitoring for detecting significant arrhythmias in patients with palpitations. Methods: This was a single-center prospective trial with 58 participants who presented with palpitations, chest pain or syncope. Outcomes were defined as the detection of any one of six arrhythmias, including supraventricular tachycardia (SVT), atrial fibrillation or atrial flutter lasting more than 30 s, pauses of more than 3 s, high-degree atrioventricular block, ventricular tachycardia (VT) >3 beats, or polymorphic VT/ventricular fibrillation. The McNemar test for paired proportions was used to compare arrhythmia detection rates. Results: The overall arrhythmia detection rate was higher with 7-day ECG patch monitoring than with 24-h Holter monitoring (34.5% vs. 19.0%, p = .008). Compared with the use of 24-h Holter monitors, the use of 7-day ECG patch monitors was associated with higher detection of SVT (29.3% vs. 13.8%, p = .042). No serious adverse skin reactions were reported among the ECG patch-monitored participants. Conclusions: The results suggest that a 7-day patch-type continuous ECG monitor is more effective for the detection of supraventricular tachycardia than is a 24-h Holter monitor. However, the clinical significance of device detected arrhythmia should be consolidated.

Translocation of cytosolic human Cdc73 to stress granules plays a role in arsenic stress-induced stabilization of p53 mRNA.

Cells trigger the assembly of stress granules (SGs) under various stress conditions. Among the many proteins recruited to SGs are RNA-binding proteins and transcription regulators. Here, we report the translocation of human (h)Cdc73, a component of the PAF1 transcription complex, to cytosolic SGs in response to arsenic stress. The hCdc73 protein possesses a long intrinsically disordered region (IDR) from amino acids 256-416, the presence of which is required for the translocation of hCdc73 to cytosolic SGs. The purified hCdc73 IDR formed droplets in vitro, and the light-activated assembly of hCdc73-IDR-mCherry-CRY2 was verified. For translocation of hCdc73 to SGs, physical interactions with SG carrier proteins, such as FMR1, are also needed. Previously, we reported that the cytosolic hCdc73-eEF1Bγ complex controls the stability of p53 mRNA. Under arsenic stress, selective sequestration of cytosolic hCdc73, but not eEF1Bγ (EEF1G) or p53 (TP53) mRNA, was detected. As a result, a transient increase in p53 mRNA at the post-transcriptional level was observed. In conclusion, we propose that the availability of mRNAs for stress-responsive genes can be controlled by restraining their negative regulators within SGs.

SHISA5/SCOTIN restrains spontaneous autophagy induction by blocking contact between the ERES and phagophores.

ABBREVIATIONS: ATG2: autophagy related 2; BECN1: beclin 1; COPII: coat protein II; DMSO: dimethyl sulfoxide; EBSS: Earle's balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERES: ER exit site(s); GFP: green fluorescent protein; H89: H-89 dihydrochloride hydrate; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NS5A: nonstructural protein 5A; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLA: proximity ligation assay; PtdIns3P: phosphatidylionositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SBP: streptavidin binding protein; SEC16A: SEC16 homolog A, endoplasmic reticulum export factor; SEC31A: SEC31 homolog A, COPII coat complex component; siRNA: small interfering RNA; Str: streptavidin; ULK1: unc-51-like autophagy activating kinase 1; VSVG: vesicular stomatitis virus glycoprotein; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.

MON-2, a Golgi protein, mediates autophagy-dependent longevity in Caenorhabditis elegans.

The Golgi apparatus plays a central role in trafficking cargoes such as proteins and lipids. Defects in the Golgi apparatus lead to various diseases, but its role in organismal longevity is largely unknown. Using a quantitative proteomic approach, we found that a Golgi protein, MON-2, was up-regulated in long-lived Caenorhabditis elegans mutants with mitochondrial respiration defects and was required for their longevity. Similarly, we showed that DOP1/PAD-1, which acts with MON-2 to traffic macromolecules between the Golgi and endosome, contributed to the longevity of respiration mutants. Furthermore, we demonstrated that MON-2 was required for up-regulation of autophagy, a longevity-associated recycling process, by activating the Atg8 ortholog GABARAP/LGG-1 in C. elegans. Consistently, we showed that mammalian MON2 activated GABARAPL2 through physical interaction, which increased autophagic flux in mammalian cells. Thus, the evolutionarily conserved role of MON2 in trafficking between the Golgi and endosome is an integral part of autophagy-mediated longevity.

Homogenization of a reaction diffusion equation can explain influenza A virus load data.

We study the influence of spatial heterogeneity on the antiviral activity of mouse embryonic fibroblasts (MEF) infected with influenza A. MEF of type Ube1L-/- are composed of two distinct sub-populations, the strong type that sustains a strong viral infection and the weak type, sustaining a weak viral load. We present new data on the virus load infection of Ube1L-/-, which have been micro-printed in a checker board pattern of different sizes of the inner squares. Surprisingly, the total viral load at one day after inoculation significantly depends on the sizes of the inner squares. We explain this observation by using a reaction diffusion model and we show that mathematical homogenization can explain the observed inhomogeneities. If the individual patches are large, then the growth rate and the carrying capacity will be the arithmetic means of the patches. For finer and finer patches the average growth rate is still the arithmetic mean, however, the carrying capacity uses the harmonic mean. While fitting the PDE to the experimental data, we also predict that a discrepancy in virus load would be unobservable after only half a day. Furthermore, we predict the viral load in different inner squares that had not been measured in our experiment and the travelling distance the virions can reach after one day.

All-Inkjet-Printed 3D Alveolar Barrier Model with Physiologically Relevant Microarchitecture.

With the outbreak of new respiratory viruses and high mortality rates of pulmonary diseases, physiologically relevant models of human respiratory system are urgently needed to study disease pathogenesis, drug efficacy, and pharmaceutics. In this paper, a 3D alveolar barrier model fabricated by printing four human alveolar cell lines, namely, type I and II alveolar cells (NCI-H1703 and NCI-H441), lung fibroblasts (MRC5), and lung microvascular endothelial cells (HULEC-5a) is presented. Automated high-resolution deposition of alveolar cells by drop-on-demand inkjet printing enables to fabricate a three-layered alveolar barrier model with an unprecedented thickness of ≈10 µm. The results show that the 3D structured model better recapitulate the structure, morphologies, and functions of the lung tissue, compared not only to a conventional 2D cell culture model, as expected, but also a 3D non-structured model of a homogeneous mixture of the alveolar cells and collagen. Finally, it is demonstrated that this thin multilayered model reproduce practical tissue-level responses to influenza infection. Drop-on-demand inkjet-printing is an enabling technology for customization, scalable manufacturing, and standardization of their size and growth, and it is believed that this 3D alveolar barrier model can be used as an alternative to traditional test models for pathological and pharmaceutical applications.

Author Correction: Freeform micropatterning of living cells into cell culture medium using direct inkjet printing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MTFMT deficiency correlates with reduced mitochondrial integrity and enhanced host susceptibility to intracellular infection.

Mitochondria behave as functional and structural hubs for innate defense against intracellular infection. While the mitochondrial membrane serves as a platform for the assembly of signaling complexes activated by intracellular infection, various danger molecules derived from impaired mitochondria activate innate signaling pathways. Using methionyl-tRNA formyl transferase (MTFMT)-deficient cells, which exhibit impaired mitochondrial activity, we examined the role of mitochondrial integrity in regulating innate defense against infection. Since MTFMT functions at the early steps of mitochondrial translation, its loss was expected to cause defects in mitochondrial activity. Under transient MTFMT gene silencing conditions, we observed shortened mitochondria along with reduced activity. MTFMT-silenced cells were more susceptible to intracellular infection, as examined by infection with RNA viruses and the intracellular bacterium Shigella flexneri. In support of this observation, MTFMT-silenced cells possessed lowered basal NF-κB activity, which remained low after S. flexneri infection. In addition, the mitochondrial accumulation of evolutionarily conserved signaling intermediate in Toll pathway (ECSIT), an adaptor protein for NF-κB activation, was significantly decreased in MTFMT-silenced cells, explaining the reduced NF-κB activity observed in these cells. Since impaired mitochondria likely release mitochondrial molecules, we evaluated the contribution of mitochondrial N-formyl peptides to the regulation of bacterial infection. Transient transfection of mitochondrial-derived N-formyl peptides favored S. flexneri infection, which was accompanied by enhanced bacterial survival, but did not affect host cell viability. However, transient transfection of mitochondrial-derived N-formyl peptides did not affect basal NF-κB activity. Altogether, these data suggest that the integrity of mitochondria is essential to their proper function in protecting against infection, as intact mitochondria not only block the release of danger molecules but also serve as signaling hubs for the downstream NF-κB pathway.

Inhibition of the oligosaccharyl transferase in Caenorhabditis elegans that compromises ER proteostasis suppresses p38-dependent protection against pathogenic bacteria.

The oligosaccharyl transferase (OST) protein complex mediates the N-linked glycosylation of substrate proteins in the endoplasmic reticulum (ER), which regulates stability, activity, and localization of its substrates. Although many OST substrate proteins have been identified, the physiological role of the OST complex remains incompletely understood. Here we show that the OST complex in C. elegans is crucial for ER protein homeostasis and defense against infection with pathogenic bacteria Pseudomonas aeruginosa (PA14), via immune-regulatory PMK-1/p38 MAP kinase. We found that genetic inhibition of the OST complex impaired protein processing in the ER, which in turn up-regulated ER unfolded protein response (UPRER). We identified vitellogenin VIT-6 as an OST-dependent glycosylated protein, critical for maintaining survival on PA14. We also showed that the OST complex was required for up-regulation of PMK-1 signaling upon infection with PA14. Our study demonstrates that an evolutionarily conserved OST complex, crucial for ER homeostasis, regulates host defense mechanisms against pathogenic bacteria.

The Interferon-Inducible Proteoglycan Testican-2/SPOCK2 Functions as a Protective Barrier against Virus Infection of Lung Epithelial Cells.

Proteoglycans function not only as structural components of the extracellular compartment but also as regulators of various cellular events, including cell migration, inflammation, and infection. Many microbial pathogens utilize proteoglycans to facilitate adhesion and invasion into host cells. Here we report a secreted form of a novel heparan sulfate proteoglycan that functions against virus infection. The expression of SPOCK2/testican-2 was significantly induced in virus-infected lungs or in interferon (IFN)-treated alveolar lung epithelial cells. Overexpression from a SPOCK2 expression plasmid alone or the treatment of cells with recombinant SPOCK2 protein efficiently blocked influenza virus infection at the step of viral attachment to the host cell and entry. Moreover, mice treated with purified SPOCK2 were protected against virus infection. Sialylated glycans and heparan sulfate chains covalently attached to the SPOCK2 core protein were critical for its antiviral activity. Neuraminidase (NA) of influenza virus cleaves the sialylated moiety of SPOCK2, thereby blocking its binding to the virus. Our data suggest that IFN-induced SPOCK2 functions as a decoy receptor to bind and block influenza virus infection, thereby restricting entry of the infecting virus into neighboring cells.IMPORTANCE Here we report a novel proteoglycan protein, testican-2/SPOCK2, that prevents influenza virus infection. Testican-2/SPOCK2 is a complex type of secreted proteoglycan with heparan sulfate GAG chains attached to the core protein. SPOCK2 expression is induced upon virus infection or by interferons, and the protein is secreted to an extracellular compartment, where it acts directly to block virus-cell attachment and entry. Treatment with purified testican-2/SPOCK2 protein can efficiently block influenza virus infection in vitro and in vivo We also identified the heparan sulfate moiety as a key regulatory module for this inhibitory effect. Based on its mode of action (cell attachment/entry blocker) and site of action (extracellular compartment), we propose testican-2/SPOCK2 as a potential antiviral agent that can efficiently control influenza virus infection.

Pathogenicity of the H1N1 influenza virus enhanced by functional synergy between the NPV100I and NAD248N pair.

By comparing and measuring covariations of viral protein sequences from isolates of the 2009 pH1N1 influenza A virus (IAV), specific substitutions that co-occur in the NP-NA pair were identified. To investigate the effect of these co-occurring substitution pairs, the V100I substitution in NP and the D248N substitution in NA were introduced into laboratory-adapted WSN IAVs. The recombinant WSN with the covarying NPV100I-NAD248N pair exhibited enhanced pathogenicity, as characterized by increased viral production, increased death and inflammation of host cells, and high mortality in infected mice. Although direct interactions between the NPV100I and NAD248N proteins were not detected, the RNA-binding ability of NPV100I was increased, which was further strengthened by NAD248N, in expression-plasmid-transfected cells. Additionally, the NAD248N protein was frequently recruited within lipid rafts, indirectly affecting the RNA-binding ability of NP as well as viral release. Altogether, our data indicate that the covarying NPV100I-NAD248N pair obtained from 2009 pH1N1 IAV sequence information function together to synergistically augment viral assembly and release, which may explain the observed enhanced viral pathogenicity.

KIN-4/MAST kinase promotes PTEN-mediated longevity of Caenorhabditis elegans via binding through a PDZ domain.

PDZ domain-containing proteins (PDZ proteins) act as scaffolds for protein-protein interactions and are crucial for a variety of signal transduction processes. However, the role of PDZ proteins in organismal lifespan and aging remains poorly understood. Here, we demonstrate that KIN-4, a PDZ domain-containing microtubule-associated serine-threonine (MAST) protein kinase, is a key longevity factor acting through binding PTEN phosphatase in Caenorhabditis elegans. Through a targeted genetic screen for PDZ proteins, we find that kin-4 is required for the long lifespan of daf-2/insulin/IGF-1 receptor mutants. We then show that neurons are crucial tissues for the longevity-promoting role of kin-4. We find that the PDZ domain of KIN-4 binds PTEN, a key factor for the longevity of daf-2 mutants. Moreover, the interaction between KIN-4 and PTEN is essential for the extended lifespan of daf-2 mutants. As many aspects of lifespan regulation in C. elegans are evolutionarily conserved, MAST family kinases may regulate aging and/or age-related diseases in mammals through their interaction with PTEN.

Transcriptional modulation of the T helper 17/interleukin 17 axis ameliorates renal ischemia-reperfusion injury.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor critical for T-cell function. Although inhibition of the Janus kinase 2 (JAK2)/STAT3 pathway has been reported to be protective against ischemia-reperfusion injury (IRI), the role of T cell-associated STAT3 in the pathogenesis of renal IRI has not been specifically defined. METHODS: We induced renal IRI in both mice with T cell-specific STAT3 knockout (Lck-Cre;STAT3flox/flox) and wild-type controls (C57BL/6) and assessed renal damage and inflammation at 48 h after IRI. Human proximal tubular epithelial cells grown under hypoxia were treated with a JAK2 inhibitor, caffeic acid 3,4-dihydroxy-phenylethyl ester, to determine the effect of JAK2/STAT3 inhibition on renal epithelia. Independently, we disrupted Cln 3-requiring 9 (Ctr9) to inhibit T helper 17 (Th17) activation via RNA interference and determined if Ctr9 inhibition aggravates renal injury through upregulated Th17 activation. RESULTS: The Lck-Cre;STAT3flox/flox mice exhibited significantly reduced kidney damage compared with controls. This protective effect was associated with reduced intrarenal Th17 infiltration and proinflammatory cytokines. Human proximal tubular epithelial cells under hypoxia exhibited significant upregulation of interleukin 17 receptors, and pharmacologic inhibition of JAK2 significantly ameliorated this change. RNA interference with Ctr9 in splenocytes enhanced differentiation into Th17 cells. In vivo knockdown of Ctr9 in mice with renal IRI further aggravated Th17-associated inflammation and kidney injury. CONCLUSIONS: STAT3 in T cells contributes to renal IRI through Th17 activation. Inhibition of Ctr9 further enhances Th17 activation and aggravates kidney injury, further supporting the role of Th17 cells in renal IRI.

Formyl-methionine as an N-degron of a eukaryotic N-end rule pathway.

In bacteria, nascent proteins bear the pretranslationally generated N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet of bacterial proteins is a degradation signal, termed fMet/N-degron. By contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were presumed to bear unformylated Nt-Met. Here we found that the yeast formyltransferase Fmt1, although imported into mitochondria, could also produce Nt-formylated proteins in the cytosol. Nt-formylated proteins were strongly up-regulated in stationary phase or upon starvation for specific amino acids. This up-regulation strictly required the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins act as fMet/N-degrons and identified the Psh1 ubiquitin ligase as the recognition component of the eukaryotic fMet/N-end rule pathway, which destroys Nt-formylated proteins.

Publisher Correction: Freeform micropatterning of living cells into cell culture medium using direct inkjet printing.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Freeform micropatterning of living cells into cell culture medium using direct inkjet printing.

Microfabrication methods have widely been used to control the local cellular environment on a micron scale. However, accurately mimicking the complexity of the in vivo tissue architecture while maintaining the freedom of form and design is still a challenge when co-culturing multiple types of cells on the same substrate. For the first time, we present a drop-on-demand inkjet printing method to directly pattern living cells into a cell-friendly liquid environment. High-resolution control of cell location is achieved by precisely optimizing printing parameters with high-speed imaging of cell jetting and impacting behaviors. We demonstrated the capabilities of the direct cell printing method by co-printing different cells into various designs, including complex gradient arrangements. Finally, we applied this technique to investigate the influence of the heterogeneity and geometry of the cell population on the infectivity of seasonal H1N1 influenza virus (PR8) by generating A549 and HeLa cells printed in checkboard patterns of different sizes in a medium-filled culture dish. Direct inkjet cell patterning can be a powerful and versatile tool for both fundamental biology and applied biotechnology.

Single-cell analysis of early antiviral gene expression reveals a determinant of stochastic IFNB1 expression.

RIG-I-like receptors (RLRs) are cytoplasmic sensors of viral RNA that trigger the signaling cascade that leads to type I interferon (IFN) production. Transcriptional induction of RLRs by IFN is believed to play the role of positive feedback to further amplify viral sensing. We found that RLRs and several other IFN-stimulated genes (ISGs) are induced early in viral infection independent of IFN. Expression of these early ISGs requires IRF3/IRF7 and is highly correlated amongst them. Simultaneous detection of mRNA of IFNB1, viral replicase, and ISGs revealed distinct populations of IFNB1 expressing and non-expressing cells which are highly correlated with the levels of early ISGs but are uncorrelated with IFN-dependent ISGs and viral gene expression. Individual expression of RLRs made IFNB1 expression more robust and earlier, suggesting a causal relation between levels of RLR and induction of IFN.

Mitochondrial chaperone HSP-60 regulates anti-bacterial immunity via p38 MAP kinase signaling.

Mitochondria play key roles in cellular immunity. How mitochondria contribute to organismal immunity remains poorly understood. Here, we show that HSP-60/HSPD1, a major mitochondrial chaperone, boosts anti-bacterial immunity through the up-regulation of p38 MAP kinase signaling. We first identify 16 evolutionarily conserved mitochondrial components that affect the immunity of Caenorhabditis elegans against pathogenic Pseudomonas aeruginosa (PA14). Among them, the mitochondrial chaperone HSP-60 is necessary and sufficient to increase resistance to PA14. We show that HSP-60 in the intestine and neurons is crucial for the resistance to PA14. We then find that p38 MAP kinase signaling, an evolutionarily conserved anti-bacterial immune pathway, is down-regulated by genetic inhibition of hsp-60, and up-regulated by increased expression of hsp-60 Overexpression of HSPD1, the mammalian ortholog of hsp-60, increases p38 MAP kinase activity in human cells, suggesting an evolutionarily conserved mechanism. Further, cytosol-localized HSP-60 physically binds and stabilizes SEK-1/MAP kinase kinase 3, which in turn up-regulates p38 MAP kinase and increases immunity. Our study suggests that mitochondrial chaperones protect host eukaryotes from pathogenic bacteria by up-regulating cytosolic p38 MAPK signaling.