Role of the novel endoribonuclease SLFN14 and its disease-causing mutations in ribosomal degradation.

Platelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by a low platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants colocalize with ribosomes and mediate rRNA endonucleolytic degradation. Compared to SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients' platelets. Overall, we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.

Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction.

In addition to mutations in ITG2B or ITGB3 genes that cause defective αIIbß3 expression and/or function in Glanzmann's thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate inside-out activation of αIIbß3 The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin (family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In 2 Spanish siblings from family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2 CalDAG-GEFI expression was markedly reduced in platelets from all patients, and by using a novel in vitro assay, we found that the nucleotide exchange activity was dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in αIIbß3 integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading, and clot retraction were not markedly affected. Integrin activation in the patients' neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation.

Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects.

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified "pathogenic" or "likely pathogenic" variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.

Inherited thrombocytopenia: novel insights into megakaryocyte maturation, proplatelet formation and platelet lifespan.

The study of patients with inherited bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets and their precursor, the megakaryocyte. The normal range of platelet counts in the bloodstream ranges from 150 000 to 400 000 platelets per microliter and is normally maintained within a narrow range for each individual. This requires a constant balance between thrombopoiesis, which is primarily controlled by the cytokine thrombopoietin (TPO), and platelet senescence and consumption. Thrombocytopenia can be defined as a platelet count of less than 150 000 per microliter and can be acquired or inherited. Heritable forms of thrombocytopenia are caused by mutations in genes involved in megakaryocyte differentiation, platelet production and platelet removal. In this review, we will discuss the main causative genes known for inherited thrombocytopenia and highlight their diverse functions and whether these give clues on the processes of platelet production, platelet function and platelet lifespan. Additionally, we will highlight the recent advances in novel genes identified for inherited thrombocytopenia and their suggested function.

Tight junction regulation through vesicle trafficking: bringing cells together.

Epithelial layers are integral for many physiological processes and are maintained by intercellular adhesive structures. During disease, these structures can disassemble, leading to breakdown of epithelia. TJs (tight junctions) are one type of intercellular adhesion. Loss of TJs has been linked to the pathogenesis of many diseases. The present review focuses on the role of vesicle trafficking in regulation of TJs, in particular trafficking of the TJ protein occludin. We examine how endocytosis and endosomal recycling modulate occludin localization under steady-state conditions and during stimulated TJ disassembly.

Hepatitis C virus induces CD81 and claudin-1 endocytosis.

Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.

Clathrin-mediated endocytosis regulates occludin, and not focal adhesion, distribution during epithelial wound healing.

BACKGROUND INFORMATION: Vesicle trafficking has long been suggested to play mechanistic roles in regulating directed cell migration. Recent evidence demonstrates that specific cell types and modes of migration involve transport of particular cargo through particular pathways. Epithelial wound healing is essential in tissue repair. However, investigations into the mechanisms regulating cell migration have mainly focused upon other models such as fibroblast-derived cells. Roles for vesicle trafficking pathways in regulating directed cell migration have been identified in recent studies, but mechanisms through which endocytosis might be involved in epithelial wound healing have not been as well studied. Therefore, we analysed potential regulatory roles for endocytosis pathways during epithelial cell motility, with a particular focus on cell adhesion. RESULTS: Specifically, and in contrast to studies in fibroblasts, we find no evidence for a link between endocytosis and the distribution of focal adhesions. However, the localisation of occludin, an essential component of tight junctions, is regulated through endocytosis. We identified epithelial monolayer wounding as a stimulus for endocytosis of occludin and have shown that internalisation of occludin from the wound edge occurs through clathrin-mediated endocytosis (CME) into a rab5-positive compartment. CONCLUSIONS: Thus, these studies have evaluated mechanistic roles for dynamin-dependant, CME and caveolar endocytosis during epithelial wound healing and have provided contrasting observations between analyses of cell motility in fibroblast models and epithelial cells. In conclusion, these studies have identified a novel mechanism for regulation of occludin during wound healing.

The role of vesicle trafficking in epithelial cell motility.

Cell motility is important for many physiological and pathological processes including organ development, wound healing, cancer metastasis and correct immune responses. In particular, epithelial wound healing is both a medically relevant topic and a common experimental model. Mechanisms underlying generation of a polarized cell and maintenance of a motile phenotype during steady-state migration are not well understood. Polarized trafficking of bulk membrane and cell adhesion molecules has been implicated in regulation of cell motility. The present review focuses on the role of different trafficking pathways in epithelial cell migration, including clathrin-mediated endocytosis, caveolar endocytosis, exocytosis of biosynthetic cargo, 'short-loop' and 'long-loop' endosomal recycling.

Schlafen 14 (SLFN14) is a novel antiviral factor involved in the control of viral replication.

Schlafen (SLFN) proteins have been suggested to play important functions in cell proliferation and immune cell development. In this study, we determined the antiviral activities of putative RNA-helicase domain-containing SLFN14. Murine SLFN14 expression was specifically induced by TLR3-mediated pathways and type I interferon (IFN) in RAW264.7 mouse macrophages. To examine the role of SLFN during viral infection, cells were infected with either wild-type PR8 or delNS1/PR8 virus. SLFN14 expression was specifically induced following influenza virus infection. Overexpression of SLFN14 in A549 cells reduced viral replication, whereas knockdown of SLFN14 in RAW264.7 cells enhanced viral titers. Furthermore, SLFN14 promoted the delay in viral NP translocation from cytoplasm to nucleus and enhanced RIG-I-mediated IFN-ß signaling. In addition, SLFN14 overexpression promoted antiviral activity against varicella zoster virus (VZV), a DNA virus. In conclusion, our data suggest that SLFN14 is a novel antiviral factor for both DNA and RNA viruses.

A Carotenoid-Deficient Mutant in Pantoea sp. YR343, a Bacteria Isolated from the Rhizosphere of Populus deltoides, Is Defective in Root Colonization.

The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid (IAA). Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of IAA. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343.

SLFN14 mutations underlie thrombocytopenia with excessive bleeding and platelet secretion defects.

Inherited thrombocytopenias are a group of disorders that are characterized by a low platelet count and are sometimes associated with excessive bleeding that ranges from mild to severe. We evaluated 36 unrelated patients and 17 family members displaying thrombocytopenia that were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study. All patients had a history of excessive bleeding of unknown etiology. We performed platelet phenotyping and whole-exome sequencing (WES) on all patients and identified mutations in schlafen 14 (SLFN14) in 12 patients from 3 unrelated families. Patients harboring SLFN14 mutations displayed an analogous phenotype that consisted of moderate thrombocytopenia, enlarged platelets, decreased ATP secretion, and a dominant inheritance pattern. Three heterozygous missense mutations were identified in affected family members and predicted to encode substitutions (K218E, K219N, and V220D) within an ATPase-AAA-4, GTP/ATP-binding region of SLFN14. Endogenous SLFN14 expression was reduced in platelets from all patients, and mutant SLFN14 expression was markedly decreased compared with that of WT SLFN14 when overexpressed in transfected cells. Electron microscopy revealed a reduced number of dense granules in affected patients platelets, correlating with a decreased ATP secretion observed in lumiaggregometry studies. These results identify SLFN14 mutations as cause for an inherited thrombocytopenia with excessive bleeding, outlining a fundamental role for SLFN14 in platelet formation and function.

Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

Tight junctions (TJs) link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK) epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes). By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor.

Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.

Transgenic mice overexpressing Renin exhibit glucose intolerance and diet-genotype interactions.

Numerous animal and clinical investigations have pointed to a potential role of the renin-angiotensin system (RAS) in the development of insulin resistance and diabetes in conditions of expanded fat mass. However, the mechanisms underlying this association remain unclear. We used a transgenic mouse model overexpressing renin in the liver (RenTgMK) to examine the effects of chronic activation of RAS on adiposity and insulin sensitivity. Hepatic overexpression of renin resulted in constitutively elevated plasma angiotensin II (four- to six-fold increase vs. wild-type, WT). Surprisingly, RenTgMK mice developed glucose intolerance despite low levels of adiposity and insulinemia. The transgenics also had lower plasma triglyceride levels. Glucose intolerance in transgenic mice fed a low-fat diet was comparable to that observed in high-fat fed WT mice. These studies demonstrate that overexpression of renin and associated hyperangiotensinemia impair glucose tolerance in a diet-dependent manner and further support a consistent role of RAS in the pathogenesis of diabetes and insulin resistance, independent of changes in fat mass.

Moving forward: polarised trafficking in cell migration.

Polarised vesicle trafficking has been suggested to regulate cell migration. Understanding how this takes place has been complicated by the use of disparate assays and cellular models. Although polarised trafficking does occur in cell motility it is not clear which pathways are involved. We propose a model for migrating cells where caveolar endocytosis occurs at the rear of the adherent surface, whereas clathrin-mediated endocytosis takes place in the middle-to-front region of the cell. We also suggest there is evidence to support polarised recycling of internalised cargo to the leading edge of migrating cells. Further research is required to confirm our hypothesis and a systematic evaluation of multiple pathways within individual systems and across different models is needed.