The retromer complex regulates C. elegans development and mammalian ciliogenesis.

The mammalian retromer consists of subunits VPS26 (either VPS26A or VPS26B), VPS29 and VPS35, and a loosely associated sorting nexin (SNX) heterodimer or a variety of other SNX proteins. Despite involvement in yeast and mammalian cell trafficking, the role of retromer in development is poorly understood, and its impact on primary ciliogenesis remains unknown. Using CRISPR/Cas9 editing, we demonstrate that vps-26-knockout worms have reduced brood sizes, impaired vulval development and decreased body length, all of which have been linked to ciliogenesis defects. Although preliminary studies did not identify worm ciliary defects, and impaired development limited additional ciliogenesis studies, we turned to mammalian cells to investigate the role of retromer in ciliogenesis. VPS35 localized to the primary cilium of mammalian cells, and depletion of VPS26, VPS35, VPS29, SNX1, SNX2, SNX5 or SNX27 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the centriolar protein, CP110 (also known as CCP110), and was required for its removal from the mother centriole. Herein, we characterize new roles for retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, suggesting a novel role for retromer in CP110 removal from the mother centriole.

Characterization of the First Secreted Sorting Nexin Identified in the Leishmania Protists.

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.

Cancer-associated fibroblast-derived exosome miR-181b-3p promotes the occurrence and development of colorectal cancer by regulating SNX2 expression.

Tumor microenvironment (TME) (e.g., stromal cells) has been closely related to the pathological process of colorectal cancer (CRC). In TME, tumor-associated fibroblasts (CAFs) are the main stromal cells. The studies have showed that CAFs promoted tumor growth and metastasis in CRC and led to poor prognosis. Mounting evidence indicates that CAFs-mediated exosomes regulate the pathological process of neighboring tumor cells through the transmission of miRNAs. In our study, we aimed to explore the function of CAFs-derived exosome miR-181b-3p in CRC. First, the expression of miR-181b-3p in CRC was found to be up-regulated and its expression was dramatically up-regulated in CRC cells after co-incubation of CAFs-mediated exosomes with CRC cells. Then, it was found that the CAFs-derived exosomes were markedly enhanced the proliferation and migration of the CRC cells, and substantially reduced apoptosis. To elucidate the influence of CAFs-derived exosome miR-181b-3p on CRC, we overexpressed and knocked down the miR-181b-3p expression in CAFs, respectively. It was found that miR-181b-3p significantly increased the proliferation and migration of CRC cells. Furthermore, we conducted in vivo experiments. Finally, we demonstrated that CAF-derived exosome miR-181b-3p regulated sorting nexin 2 (SNX2) expression in CRC cells by bioinformatics prediction combined with luciferase reporter assay. Further cellular and animal experiments jointly elucidated that miR-181b-3p promoted the pathological process of CRC by SNX2 expression. In brief, our results demonstrated that CAFs-derived exosome miR-181b-3p promoted the pathogenesis of CRC by regulating SNX2 expression, which provides a novel idea for CRC treatment.

Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication.

BACKGROUND: Replication of the influenza A viruses occurs in the cells through the viral RdRP consisting of PB1, PB2, and PA. Several cellular proteins are involved in these processes. This study aims to reveal the interaction between human SNX2 protein and the PA protein and the effects of the SNX2 on the virus replication. RESULTS: To identify potential host interacting proteins to the PA, yeast two-hybrid assay was carried out with HEK293 cell cDNA library and the PA as a bait. We focused on SNX2 protein, which interacts with the PA in the yeast cells. By using the co-immunoprecipitation assays, it has been demonstrated that the amino-terminal part of the PA was important for binding to the SNX2. Immunolocalization of the proteins in HeLa cells supported this interaction. Knockdown of the SNX2 with siRNA in the cells resulted in a significant increase in both viral transcripts and virus growth. However, the increase of SNX2 in transfected cells didn't cause a significant change in the viral RdRP activity in minireplicon assay. This may suggest that the negative effect of SNX2 on the virus replication could be saturated with its authentic intra-cellular amount. CONCLUSIONS: This study revealed that the SNX2 and PA protein interact with each other in both yeast and HEK293 cells, and the SNX2 has a negative regulatory function on the virus replication. However, more knowledge is required to elucidate the action mechanism of the SNX2 on the influenza A virus replication at the molecular level.

Diacylglycerol kinase and phospholipase D inhibitors alter the cellular lipidome and endosomal sorting towards the Golgi apparatus.

The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.

Poor responses to tyrosine kinase inhibitors in a child with precursor B-cell acute lymphoblastic leukemia with SNX2-ABL1 chimeric transcript.

In addition to BCR, various rare fusion partners for the ABL1 gene have been reported in leukemia. We have identified the fusion gene SNX2-ABL1 in a pediatric case of acute lymphoblastic leukemia (ALL), which has only once previously been reported in an adult patient. Cytogenetic analysis detected this fusion gene arising from a t(5;9)(q22;q34) translocation. ALL cells carrying a SNX2-ABL1 fusion exhibited a BCR-ABL1+ ALL-like gene expression profile. The patient poorly responded to dasatinib but partially responded to imatinib. Treatment using tyrosine kinase inhibitors requires further investigation to optimize the genotype-based treatment stratification for patients with SNX2-ABL1 fusion.

Sensitivity of SNX2-ABL1 toward tyrosine kinase inhibitors distinct from that of BCR-ABL1.

We introduced SNX2-ABL1, a novel ABL1-related chimeric transcript lacks SH3 and SH2 domains, into murine Ba/F3 cells and compared their function with that of BCR-ABL1. After the expression of SNX2-ABL1 proteins, Ba/F3 cells acquired an ability to proliferate in an IL-3-independent manner. Upon treatment with both imatinib and dasatinib, BCR-ABL1-expressing Ba/F3 cells underwent rapid apoptosis, whereas SNX2-ABL1-expressing Ba/F3 cells showed poorer sensitivity toward these TKIs and could proliferate in the presence of a low dose of dasatinib. Therefore, other TKIs with a more selective effect against this chimeric kinase should be used for the treatment of patients with SNX2-ABL1+ ALL.