Lymphocyte Cytosolic Protein 1 (L-plastin) I232F Mutation Impairs Granulocytic Proliferation and Causes Neutropenia

Neutrophils migrate into inflamed tissue, engage in phagocytosis, and clear pathogens or apoptotic cells. These processes require well-coordinated events involving the actin cytoskeleton. We describe a child with severe neutropenia and episodes of soft tissue infections and pneumonia. Bone marrow examination showed granulocytic hypoplasia with dysplasia. Whole exome sequencing revealed a de novo heterozygous missense mutation in LCP1, which encodes the F-actin binding protein Lymphocyte Cytosolic Protein 1. To determine its pathophysiologic significance, we stably transduced cells with a doxycycline-inducible wild type LCP1 and LCP1 I232F lentiviral constructs. We observed dysplastic granulocytic 32D cells expressing LCP1 I232F cells. These cells showed decreased proliferation without a block in differentiation. Additionally, expression of LCP1 I232F resulted in a cell cycle arrest at G2/M phase, but it did not lead to increased levels of genes involved in apoptosis or the unfolded protein response. Both 32D and HeLa cells expressing mutant LCP1 showed impaired cell motility and invasiveness. Flow cytometry showed increased F-actin. However, mutant LCP1-expressing 32D cells demonstrated normal oxidative burst upon stimulation. Confocal imaging and subcellular fractionation revealed diffuse intracellular localization of LCP1, but only the mutant form was found in the nucleus. We conclude that LCP1 is a new gene involved in granulopoiesis, and the missense variant LCP1 I232F leads to neutropenia and granulocytic dysplasia with aberrant actin dynamics. Our work supports a model of neutropenia due to aberrant actin regulation.

Uncomplicated Plasmodium vivax malaria: mapping the proteome from circulating platelets

Background Thrombocytopenia is frequent in Plasmodium vivax malaria but the role of platelets in pathogenesis is unknown. Our study explores the platelet (PLT) proteome from uncomplicated P. vivax patients, to fingerprint molecular pathways related to platelet function. Plasma levels of Platelet factor 4 (PF4/CXCL4) and Von Willebrand factor (VWf), as well as in vitro PLTs—P. vivax infected erythrocytes (Pv-IEs) interactions were also evaluated to explore the PLT response and effect on parasite development. Methods A cohort of 48 patients and 25 healthy controls were enrolled. PLTs were purified from 5 patients and 5 healthy controls for Liquid Chromatography–Mass spectrometry (LC–MS/MS) analysis. Plasma levels of PF4/CXCL4 and VWf were measured in all participants. Additionally, P. vivax isolates (n = 10) were co-cultured with PLTs to measure PLT activation by PF4/CXCL4 and Pv-IE schizonts formation by light microscopy. Results The proteome from uncomplicated P. vivax patients showed 26 out of 215 proteins significantly decreased. PF4/CXCL4 was significantly decreased followed by other proteins involved in platelet activation, cytoskeletal remodeling, and endothelial adhesion, including glycoprotein V that was significantly decreased in thrombocytopenic patients. In contrast, acute phase proteins, including SERPINs and Amyloid Serum A1 were increased. High levels of VWf in plasma from patients suggested endothelial activation while PF4/CXCL4 plasma levels were similar between patients and controls. Interestingly, high levels of PF4/CXCL4 were released from PLTs—Pv-IEs co-cultures while Pv-IEs schizont formation was inhibited. Conclusions The PLT proteome analyzed in this study suggests that PLTs actively respond to P. vivax infection. Altogether, our findings suggest important roles of PF4/CXCL4 during uncomplicated P. vivax infection through a possible intracellular localization. Our study shows that platelets are active responders to P. vivax infection, inhibiting intraerythrocytic parasite development. Future studies are needed to further investigate the molecular pathways of interaction between platelet proteins found in this study and host response, which could affect parasite control as well as disease progression.

The Signal Peptide and Chaperone UNC93B1 Both Influence TLR8 Ectodomain Intracellular Endosomal Localization

Toll-like receptor 8 (TLR8) is a single-stranded RNA sensing receptor and is localized in the cellular compartments, where it encounters foreign or self-nucleic acids and activates innate and adaptive immune responses. However, the mechanism controlling intracellular localization TLR8 is not completely resolved. We previously revealed the intracellular localization of TLR8 ectodomain (ECD), and in this study, we investigated the mechanism of the intracellular localization. Here we found that TLR8 ECDs from different species as well as ECDs from different TLRs are all intracellularly localized, similarly to the full-length porcine TLR8. Furthermore, porcine, bovine, and human TLR8 ECDs are all localized in cell endosomes, reflecting the cellular localization of TLR8. Intriguingly, none of post-translational modifications at single sites, including glycosylation, phosphorylation, ubiquitination, acetylation, and palmitoylation alter porcine TLR8-ECD endosomal localization. Nevertheless, the signal peptide of porcine TLR8-ECD determines its endosomal localization. On the other hand, signaling regulator UNC93B1 also decides the endosomal localization of porcine, bovine, and human TLR8 ECDs. The results from this study shed light on the mechanisms of not only TLR8 intracellular localization but also the TLR immune signaling.

High ATF4 Expression Is Associated With Poor Prognosis, Amino Acid Metabolism, and Autophagy in Gastric Cancer

BackgroundThe role of activating transcription factor 4 (ATF4) underlying gastric cancer (GC) remains unclear. The purpose of this study was to investigate the expression levels and biological functions of ATF4 in GC.MethodsExpression of ATF4 was detected by quantitative PCR (qPCR), Western blotting, and immunohistochemistry. Cox regression was used for survival analysis and the construction of the nomogram. Immunofluorescence was used to identify the intracellular localization of ATF4. Knockdown and overexpression of ATF4 in GC cells followed by wound healing and Transwell assays, EdU and Calcein-AM/propidium iodide (PI) staining, and cell cycle detection were performed to examine its function in vitro. Transmission electron microscopy was performed to assess the autophagy levels upon ATF4 silencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA) were used to determine gene enrichment. SPSS 22.0 software, GraphPad Prism 7.0, and R version 3.6.1 were used for statistical analysis.ResultsATF4 expression was upregulated in GC cells and tissues compared with corresponding normal tissues. Survival analysis suggested that a high ATF4 expression was strongly associated with worse overall survival (OS) of GC patients (p < 0.001). The nomogram and the receiver operating characteristic (ROC) curves demonstrated that ATF4 was a highly sensitive and specific prognostic marker of GC [C-index = 0.797, area under the ROC curve (AUC) of 3-year OS = 0.855, and AUC of 5-year OS = 0.863]. In addition, ATF4 knockdown inhibited the cell proliferation, migration, invasion, and cell cycle progression of GC cells in vitro, while overexpression of ATF4 exerted the opposite effects. Bioinformatics analysis showed that ATF4 could promote GC progression possibly by regulating asparagine (Asn) metabolism and autophagy pathways. Further experiments indicated that ATF4 expression was significantly positively correlated with ASNS expression. The inhibition of cell clone formation in Asn-deprived conditions was more significant in the shATF4 group. Finally, we found that ATF4 promoted autophagy through regulating the mTORC1 pathway in GC cells.ConclusionThese findings suggested that ATF4 can significantly promote GC development and serve as an independent prognostic factor for GC.

Dynamic queuosine changes in tRNA couple nutrient levels to codon choice in Trypanosoma brucei

Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for ‘normal’ growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output.

ZO-1 Intracellular Localization Organizes Immune Response in Non-Small Cell Lung Cancer

Delocalization of zonula occludens-1 (ZO-1) from tight junctions plays a substantial role in epithelial cell plasticity observed during tumor progression. In vitro, we reported an impact of ZO-1 cyto-nuclear content in modulating the secretion of several pro-inflammatory chemokines. In vivo, we demonstrated that it promotes the recruitment of immune cells in mouse ear sponge assays. Examining lung cancers, we showed that a high density of CD8 cytotoxic T cells and Foxp3 immunosuppressive regulatory T cells in the tumor microenvironment correlated with a cyto-nuclear expression of ZO-1. Taken together, our results support that, by affecting tumor cell secretome, the cyto-nuclear ZO-1 pool may recruit immune cells, which could be permissive for tumor progression.

Retracing the evolution from antimicrobial to targeting peptides

We experimentally challenged the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert such antimicrobial peptides into bona fide organelle-targeting peptides, we expressed a set of 13 antimicrobial peptides of various origins in the green alga Chlamydomonas reinhardtii that serves as a model for both mitochondrial and chloroplast import. The peptides were modified to match distinctive features of mitochondrial and chloroplast targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as cargo protein. We present a temporal evolutionary scenario that emphasizes the early contribution of exchanging Lysines with Arginines in the sequence of the antimicrobial peptide, the evolution of a processing site followed by the addition of unstructured sequence and protein interaction sites that allow the selective targeting to the chloroplast.

Sirtuin-1 isoforms differentially regulate mitochondrial function

Alternative splicing generates multiple distinct isoforms that increase transcriptome and proteome diversity. Alternatively spliced isoforms may lose part of the protein domain and have different intracellular localization as well as distinct functions. The main form of the SIRT1 (SIRT1v1) protein contains 11 exons. We have identified two new isoforms, SIRT1v2 (lost 2 exons), and SIRT1v3 (lost 3 exons), but their effect on mitochondrial gene expression has not been reported. To study the effect of the three SIRT1 isoforms on mitochondrial gene expression and function, neuronal cells were transfected with SIRT1 isoforms v1, v2 or v3 plasmids, respectively. Gene expression was measured by quantitative reverse transcription PCR (RT-qPCR). Our data showed SIRT1 isoforms v1, v2 and v3 differentially regulated PCG-1alpha and PCG-1beta, which are the upstream regulators of mitochondrial structure and function. SIRT1v1 upregulated mitofusin-1 (MFN1), the mitochondrial dynamin-like GTPase (OPA1) gene, and the transcription factor A mitochondrial (TFAM) gene. In contrast, the SIRT1-v2 isoform repressed the MFN1, MFN2, and TFAM genes, while the SIRT1-v3 isoform repressed the MFN1 gene. In addition, the three SIRT1 isoforms differentially affected the mitochondrial respiratory complex I genes, including NDUFAB1, NDUFS1, NDUFV1, NDUFV2. The data indicates that SIRT1 regulates mitochondrial biogenesis and function through a signaling pathway involving PGC-1alpha, PCG-1beta, mitofusin 1 and 2, OPA1, and TFAM genes. Taken together, alternative splicing generated three SIRT1 isoform proteins with diverse functions. Age-related changes in the alternative splicing events are likely to impact sirtuin-regulated cellular functions and signaling pathways in aging and senescence.

TRPC3, but not TRPC1, as a good therapeutic target for standalone or complementary treatment of DMD

Background Duchenne muscular dystrophy (DMD) is an X-linked inherited disease caused by mutations in the gene encoding dystrophin that leads to a severe and ultimately life limiting muscle-wasting condition. Recombinant adeno-associated vector (rAAV)-based gene therapy is promising, but the size of the full-length dystrophin cDNA exceeds the packaging capacity of a rAAV. Alternative or complementary strategies that could treat DMD patients are thus needed. Intracellular calcium overload due to a sarcolemma permeability to calcium (SPCa) increase is an early and critical step of the DMD pathogenesis. We assessed herein whether TRPC1 and TRPC3 calcium channels may be involved in skeletal muscle SPCa alterations and could represent therapeutic targets to treat DMD. Methods All experiments were conducted in the DMDmdx rat, an animal model that closely reproduces the human DMD disease. We measured the cytosolic calcium concentration ([Ca2+]c) and SPCa in EDL (Extensor Digitorum Longus) muscle fibers from age-matched WT and DMDmdx rats of 1.5 to 7 months old. TRPC1 and TRPC3 expressions were measured in the EDL muscles at both the mRNA and protein levels, by RT-qPCR, western blot and immunocytofluorescence analysis. Results As expected from the malignant hyperthermia like episodes observed in several DMDmdx rats, calcium homeostasis alterations were confirmed by measurements of early increases in [Ca2+]c and SPCa in muscle fibers. TRPC3 and TRPC1 protein levels were increased in DMDmdx rats. This was observed as soon as 1.5 months of age for TRPC3 but only at 7 months of age for TRPC1. A slight but reliable shift of the TRPC3 apparent molecular weight was observed in DMDmdx rat muscles. Intracellular localization of both channels was not altered. We thus focused our attention on TRPC3. Application of Pyr10, a specific inhibitor of TRPC3, abolished the differences between SPCa values measured in WT and DMDmdx. Finally, we showed that a rAAV-microdystrophin based treatment induced a high microdystrophin expression but only partial prevention of calcium homeostasis alterations, skeletal muscle force and TRPC3 protein increase. Conclusions All together our results show that correcting TRPC3 channel expression and/or activity appear to be a promising approach as a single or as a rAAV-based complementary therapy to treat DMD.

Vesicular and extravesicular protein analyses from the airspaces of ozone-exposed mice revealed signatures associated with mucoinflammatory lung disease

Lung epithelial lining fluid (ELF) harbors a variety of proteins that influence homeostatic and stress responses in the airspaces. Exosomes, nano-sized extracellular vesicles, contain many proteins that vary in abundance and composition based on the prevailing conditions. Ozone causes inflammatory responses in the airspaces of experimental animals and humans. However, the exosomal protein signatures contained within the ELF from ozone-exposed lung airspaces remain poorly characterized. To explore this, we hypothesized that ozone triggers the release of exosome-bound inflammatory proteins from various cells that reflect mucoobstructive lung disease. Accordingly, we repetitively exposed adult male and female C57BL/6 mice to HEPA-filtered air (air) or 0.8 ppm ozone (4 h per day) for 14 days (five consecutive days of exposure, 2 days of rest, five consecutive days of exposure, 2 days of rest, four consecutive days of exposure). Exosome-bound proteomic signatures, as well as the levels of soluble inflammatory mediators in the bronchoalveolar lavage fluid (BALF), were determined 12–16 h after the last exposure. Principal component analyses of the exosome-bound proteome revealed a clear distinction between air-exposed and ozone-exposed mice, as well as between ozone-exposed males and ozone-exposed females. In addition to 575 proteins that were enriched in both sexes upon ozone exposure, 243 and 326 proteins were enriched uniquely in ozone-exposed males and females, respectively. Ingenuity pathway analyses on enriched proteins between ozone- and air-exposed mice revealed enrichment of pro-inflammatory pathways. More specifically, macrophage activation-related proteins were enriched in exosomes from ozone-exposed mice. Cytokine analyses on the BALF revealed elevated levels of G-CSF, KC, IP-10, IL-6, and IL-5 in ozone-exposed mice. Finally, the histopathological assessment revealed significantly enhanced intracellular localization of mucoinflammatory proteins including MUC5B and FIZZ1 in ozone-exposed mice in a cell-specific manner indicating the cellular sources of the proteins that are ferried in the exosomes upon ozone-induced lung injury. Collectively, this study identified exosomal, secretory, and cell-specific proteins and biological pathways following repetitive exposure of mice to ozone.