Intracellular Membrane Transport Research Articles

Biochar modulates intracellular electron transfer for nitrate reduction in denitrifying anaerobic methane oxidizing archaea

Denitrifying anaerobic methane oxidizing (DAMO) archaea plays a significant role in simultaneously nitrogen removal and methane mitigation, yet its limited metabolic activity hinders engineering applications. This study employed biochar to explore its potential for enhancing the metabolic activity and nitrate reduction capacity of DAMO microorganisms. Sawdust biochar (7 g/L) was found to increase the nitrate reduction rate by 2.85 times, although it did not affect the nitrite reduction rate individually. Scanning electron microscopy (SEM) and fluorescence excitation-emission matrix (EEM) analyses revealed that biochar promoted microbial aggregation, and stimulated the secretion of extracellular polymeric substances (EPS). Moreover, biochar bolstered the redox capacity and conductivity of the biofilm, notably enhancing the activity of the electron transfer system by 1.65 times. Key genes involved in intracellular electron transport (Hdr, MHC, Rnf) and membrane transport proteins (BBP, ABC, NDH) of archaea were significantly up-regulated. These findings suggest that biochar regulates electrons generated by reverse methanogenesis to the membrane for nitrate reduction.

Syntaxin6 contributes to hepatocellular carcinoma tumorigenesis via enhancing STAT3 phosphorylation

BackgroundSyntaxin6 (STX6) is a SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein complex located in the trans-Golgi network and endosomes, which is closely associated with a variety of intracellular membrane transport events. STX6 has been shown to be overexpressed in a variety of human malignant tumors such as esophageal, colorectal, and renal cell carcinomas, and participates in tumorigenesis and development.MethodsBased on clinical public database and clinical liver samples analysis, the expression of STX6 in hepatocellular carcinoma (HCC) tissues was investigated. The effects of STX6 on proliferation, migration and invasion of HCC cell in vitro and in vivo were evaluated through gain- and loss-of-function studies. We further performed RNA-seq analysis and protein interactome analysis, to further decifer the detailed mechanisms of STX6 in the regulation of the JAK-STAT pathway in HCC.ResultsSTX6 expression was upregulated in HCC tissues and its expression was highly correlated with the high histological grade of the tumor. STX6 promoted HCC cell proliferation, migration and invasion both in vitro and in vivo. Mechanistically, STX6 mediated tumor progression depending on promoting the activation of JAK-STAT signaling pathway. Receptor for activated protein kinase C (RACK1) as an essential adaptor protein mediating STX6 regulation of JAK-STAT pathway. Specifically, STX6 interacted with RACK1 and then recruited signal transducer and activator of transcription 3 (STAT3) to form a protein-binding complex and activates STAT3 transcriptional activity.ConclusionsThis study provided a novel concept that STX6 exerted oncogenic effects by activating the STAT3 signaling pathway, and STX6 might be a promising therapeutic target for HCC.

Resistance of Alicyclobacillus acidoterrestris spores to atmospheric cold plasma: Insights from sporulation temperature and mechanism analysis

Environmental conditions during sporulation, such as temperature, pH value, and mineral content, can significantly influence the formation production and resistance of spores. In this study, we investigated the sporulation capacity and resistance of Alicyclobacillus acidoterrestris (AAT) spores obtained at different temperatures (25 °C, 35 °C, 45 °C) to Dielectric Barrier Discharge Atmospheric cold plasma (DBD-ACP, 20 and 30 kV for 2–8 min). The results indicated that the efficiency of sporulation was highest at the optimum temperature (45 °C), deviation from optimal temperature would have a negative effect on sporulation. Otherwise, sporicidal efficiency of DBD-ACP increased with longer treatment time and lower sporulation temperatures, suggesting that AAT spores exhibit weaker resistance to DBD-ACP at lower sporulation temperature. Results from the release of DPA, and electron microscopic observations confirmed that DBD-ACP caused disruption of the cortex and membrane structure of AAT spores, leading to the release of intracellular contents and subsequent spore's death. Further analysis through proteomics revealed intriguing insights into the differences between spores formed at 25 °C and those at 45 °C. Specifically, spores formed at 25 °C exhibited lower expression levels of proteins related to sporulation, intracellular energy metabolism, membrane transport, and significant down-regulation of proteins involved in peptidoglycan and spore coat formation compared to those at 45 °C. These alterations likely contribute to the development of a thinner and looser spore structure, rendering it less effective in defending, i.e. less resistant to DBD-ACP treatment. Industrial relevanceAAT spores' resistance to pasteurization and persistence in NFC juices impact juice quality and safety. This study investigates the resistance of AAT spore to DBD-ACP treatment under different sporulation temperatures. Insights into sporicidal behavior of DBD-ACP and the effect of sporulation temperature on resistance provide essential knowledge for optimizing juice processing and enhancing microbial safety. The findings offer practical guidance for the juice industry to develop improved preservation strategies, ensuring longer shelf life and consumer satisfaction.

Ultrastructural Abnormalities in Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Neurons of Two Cohen Syndrome Patients.

Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

Choline chloride pretreatment on volatile fatty acids promotion from sludge anaerobic fermentation: In-situ deep eutectic solvents-like formation for EPS disintegration and associated microbial functional profiles upregulation

Slow solubilization/hydrolysis efficiency has been widely recognized as a limiting factor for volatile fatty acids (VFAs) production from waste activated sludge (WAS) via anaerobic fermentation. This study developed an innovative choline chloride (ChCl)-based approach for WAS treatment. Accordingly, the VFAs yield was increased by 2.5–623.3%, which was attributed to the concurrent acceleration of WAS solubilization, hydrolysis, and acidification. Fourier transform infrared spectrometer (FTIR) and molecular docking analysis showed that ChCl can attack and disrupt extracellular polymeric substances (EPS) by forming hydrogen bonds with organics occurred in WAS (i.e. proteins). More importantly, the generated VFAs in turn interacted with ChCl for in-situ deep eutectic solvents (DES)-like formation to significantly improve the organics dissolution of WAS for more bioavailable substrates associated with VFAs biosynthesis, and such effects were intensified with the VFAs level. Besides, ChCl exhibited “screening-effects” to enrich the hydrolytic and acidogenic anaerobes in WAS fermentation systems, while reduced VFAs-consumers. Meanwhile, the microbial metabolic profiles shift to favor the protein metabolism, and the genetic traits associated with extracellular and intracellular substrates metabolism, membrane transport, and fatty acid biosynthesis were all upregulated. Noteworthily, the functional hydrolytic and acidogenic species maintained their microbial activity for efficient VFAs production via quorum sensing (QS) and two-component systems (TCS) to counteract ChCl stress. The ranking efficiency product (REP) indices revealed that ChCl pretreatment showed a higher sustainable footprint (84.0%) over other traditional pretreatment approaches for WAS fermentation in view of both economic and environmental benefits. This work provides an innovative niche for efficient VFAs production from WAS with the sustainable application.

Intracellular Membrane Transport in Vascular Endothelial Cells.

The main component of blood and lymphatic vessels is the endothelium covering their luminal surface. It plays a significant role in many cardiovascular diseases. Tremendous progress has been made in deciphering of molecular mechanisms involved into intracellular transport. However, molecular machines are mostly characterized in vitro. It is important to adapt this knowledge to the situation existing in tissues and organs. Moreover, contradictions have accumulated within the field related to the function of endothelial cells (ECs) and their trans-endothelial pathways. This has induced necessity for the re-evaluation of several mechanisms related to the function of vascular ECs and intracellular transport and transcytosis there. Here, we analyze available data related to intracellular transport within ECs and re-examine several hypotheses about the role of different mechanisms in transcytosis across ECs. We propose a new classification of vascular endothelium and hypotheses related to the functional role of caveolae and mechanisms of lipid transport through ECs.

Syntaxin-6 promotes the progression of hepatocellular carcinoma and alters its sensitivity to chemotherapies by activating the USF2/LC3B axis.

Syntaxin-6 (STX6), a protein of the syntaxin family, is located in the trans-Golgi network and is involved in a variety of intracellular membrane transport events. STX6 is overexpressed in different human malignant tumors. However, little is known about its exact function and molecular mechanism in hepatocellular carcinoma (HCC). In this study, we found that the expression of STX6 was significantly increased in HCC tissues and was associated with poor survival. Gain- and loss-of-function experiments showed that STX6 promotes cell proliferation and metastasis of HCC cells both in vitro and in vivo. Mechanistically, STX6 was negatively regulated by the upstream stimulatory factor 2 (USF2). In addition, STX6 facilitates the association of autophagosomes with lysosomes. Importantly, we demonstrated that STX6 overexpression, despite enhanced resistance to lenvatinib, sensitizes HCC cells to the autophagy activator rapamycin. This study revealed that, under the control of USF2, STX6 accelerates the degradation of microtubule-associated protein 1 light chain 3 beta (LC3) by promoting autophagic flux, ultimately promoting HCC progression. Collectively, we suggest that the USF2-STX6-LC3B axis is a potential therapeutic target in liver cancer.

Genome-Wide Analysis of the Rab Gene Family in Melilotus albus Reveals Their Role in Salt Tolerance

Melilotus albus is a high-quality forage, due to its high protein content, and aboveground biomass and salt tolerance. Rab (Ras-related protein in the brain) proteins are the largest GTPase family which play a key role in intracellular membrane transport, and many Rab genes have been identified in eukaryotes. The growth and distribution of M. albus are severely hampered by soil salinization. However, little is known about candidate genes for salt tolerance in M. albus. In this study, 27 Rab family genes were identified for the first time from M. albus, and divided into eight groups (Groups A-H). The number of introns in MaRabs ranged from one to seven, with most genes containing one intron. In addition, most MaRab proteins showed similarities in motif composition. Phylogenetic analysis and structural-domain comparison indicated that Rab family genes were highly conserved in M. albus. Members of the MaRab gene family were distributed across all eight chromosomes, with the largest distribution on chromosome 1. Prediction of the protein interaction network showed that 24 Rab proteins exhibited protein-protein interactions. Analysis of the promoter cis-acting elements showed that MaRab-gene family members are extensively involved in abiotic stress responses. RNA-seq data analysis of the MaRab-gene-expression patterns suggested that the Rab gene family possesses differentially expressed members in five organs and under salt stress, drought stress, and ABA (Abscisic Acid) treatment. Differentially expressed genes under drought stress, salt stress and ABA stress were validated by quantitative real-time PCR. Furthermore, heterologous expression in yeast was used to characterize the functions of MaRab1 and MaRab17, which were upregulated in reaction to salt stress. In summary, this study provided valuable information for further research into the molecular mechanism of the response of M. albus to saline stress, as well as the possibility of developing cultivars with high salt-resistance characteristics.

In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis.

Subcellular fractionation is an introductory step in a variety of experimental approaches designed to study intracellular components, like membranes and organelle systems. Subcellular fractions enriched in membranes of the Golgi apparatus of mammalian cells have been isolated to address localization and activity of proteins, including enzymes, to study intracellular membrane transport mechanisms, and to reconstitute in vitro cellular processes associated with the Golgi apparatus. Here, I describe methods to purify Golgi membranes by subcellular fractionation, to assay nucleotide sulfate (PAPS) uptake into Golgi vesicles, and to measure sulfate incorporation into in vitro synthesized glycosaminoglycans.

Accessing intracellular membrane transport proteins: a novel electrophysiological approach for lysosomal and mitochondrial channels and transporters

Lysosomal, mitochondrial and other internal membranes are moving into focus of transport protein research. De-orphanization of intracellular transport proteins is ongoing and pharmacological interest is rising. For example, the lysosomal leak channel TMEM175 is investigated in the context of neurodegenerative diseases, the amino acid transporter SLC15A4 is linked to inflammatory processes and mitochondrial pumps are relevant for drug safety.

Towards epigenomic and metabolomic profiles of chronic organophosphate exposure in residents of California’ Central Valley

BACKGROUND AND AIM: Organophosphates (OP) are widely used in the agricultural Central Valley region of California. This study aimed to elucidate metabolomic and epigenetic changes induced by chronic ambient OP exposure, as well as possible interactions between different molecular layers. METHODS: We conducted high-resolution metabolomic profiling (liquid chromatography with high-resolution mass spectrometry) and genome-wide DNA methylation profiling (Illumina 450 k) of blood samples from 176 older adults living in the California Central Valley. Cumulative OP exposure from ambient sources at homes and workplaces over a ten-year period was estimated using a geographic information system (GIS)-based model. Biweighted midcorrelation (bicor) and partial least squares regression were used to identify metabolomic features and CpGs associated with OPs. Potential confounders, including age, sex, race/ethnicity, education, and cell compositions, were adjusted a prior. Functional annotation and pathway enrichment analyses were conducted for biological interpretation. We also integrated the methylome and metabolome by investigating the correlation structures existing between OP-related CpGs and metabolites and all other features via bicor. RESULTS:The single-omic analyses showed both epigenomic and metabolomic signatures of OP as being enriched in the glycosphingolipid (GSL) biosynthesis pathway. Besides this common pathway, the metabolome and epigenome also exhibited distinct responses to OPs, with differently methylated CpGs being involved in intracellular membrane transport, cell adhesion, and carcinogenesis; and OP-related metabolites being involved in aromatic amino acids metabolism, neurotransmitter precursors, oxidative stress, and mitochondria function. Moreover, we illustrate possible interactions between these two molecular layers through metabolic processes and nutrient-sensing pathways when integrating the epigenomic and metabolomic signals. CONCLUSIONS:In summary, we linked GIS-modeled chronic low-level OP pesticide data with blood metabolomic and epigenomic data in an older adult population. Our findings suggest that it seems feasible to use multi-omics integration in studies of chronic environmental exposures in humans to better understand the pathophysiology involved in pesticide-related chronic health outcomes. KEYWORDS: Organophosphates, multi-omics, metabolomics, epigenomics, glycosphingolipid

Nonlinear material and ionic transport through membrane nanotubes

Membrane nanotubes (NTs) and their networks play an important role in intracellular membrane transport and intercellular communications. The transport characteristics of the NT lumen resemble those of conventional solid-state nanopores. However, unlike the rigid pores, the soft membrane wall of the NT can be deformed by forces driving the transport through the NT lumen. This intrinsic coupling between the NT geometry and transport properties remains poorly explored. Using synchronized fluorescence microscopy and conductance measurements, we revealed that the NT shape was changed by both electric and hydrostatic forces driving the ionic and solute fluxes through the NT lumen. Far from the shape instability, the strength of the force effect is determined by the lateral membrane tension and is scaled with membrane elasticity so that the NT can be operated as a linear elastic sensor. Near shape instabilities, the transport forces triggered large-scale shape transformations, both stochastic and periodic. The periodic oscillations were coupled to a vesicle passage along the NT axis, resembling peristaltic transport. The oscillations were parametrically controlled by the electric field, making NT a highly nonlinear nanofluidic circuitry element with biological and technological implications.

Loss of MUNC18-1 leads to retrograde transport defects in neurons.

Loss of the exocytic Sec1/MUNC18 protein MUNC18‐1 or its target‐SNARE partners SNAP25 and syntaxin‐1 results in rapid, cell‐autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis‐Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether loss of MUNC18‐1 causes defects in intracellular membrane transport pathways in primary murine neurons that may explain neurodegeneration. Electron, confocal and super resolution microscopy confirmed that loss of MUNC18‐1 expression results in a smaller cis‐Golgi. In addition, we now show that medial‐Golgi and the trans‐Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall, ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. By synchronizing protein trafficking by conditional cargo retention in the endoplasmic reticulum using selective hooks (RUSH) and immunocytochemistry, we show that anterograde Endoplasmic Reticulum‐to‐Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18‐1 caused reduced retrograde Cholera Toxin B‐subunit transport from the plasma membrane to the Golgi. In addition, MUNC18‐1‐deficiency resulted in abnormalities in retrograde TrkB trafficking in an antibody uptake assay. We conclude that MUNC18‐1 deficient neurons have normal anterograde but reduced retrograde transport to the Golgi. The impairments in retrograde pathways suggest a role of MUNC18‐1 in endosomal SNARE‐dependent fusion and provide a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18‐1 deficient neurons.

The Small GTPase Superfamily in Plants: A Conserved Regulatory Module with Novel Functions

Small GTP-binding proteins represent a highly conserved signaling module in eukaryotes that regulates diverse cellular processes such as signal transduction, cytoskeletal organization and cell polarity, cell proliferation and differentiation, intracellular membrane trafficking and transport vesicle formation, and nucleocytoplasmic transport. These proteins function as molecular switches that cycle between active and inactive states, and this cycle is linked to GTP binding and hydrolysis. In this review, the roles of the plant complement of small GTP-binding proteins in these cellular processes are described, as well as accessory proteins that control their activity, and current understanding of the functions of individual members of these families in plants-with a focus on the model organism Arabidopsis-is presented. Some potential novel roles of these GTPases in plants, relative to their established roles in yeast and/or animal systems, are also discussed.

Electrophysiological Methods for Detection of Membrane Leakage and Hemifission by Dynamin 1.

Membrane fusion and fission are indispensable parts of intracellular membrane recycling and transport. Electrophysiological techniques have been instrumental in discovering and studying fusion and fission pores, the key intermediates shared by both processes. In cells, electrical admittance measurements are used to assess in real time the dynamics of the pore conductance, reflecting the nanoscale transformations of the pore, simultaneously with membrane leakage. Here, we described how this technique is adapted to in vitro mechanistic analyses of membrane fission by dynamin 1 (Dyn1), the protein orchestrating membrane fission in endocytosis. We reconstitute the fission reaction using purified Dyn1 and biomimetic lipid membrane nanotubes of defined geometry. We provide a comprehensive protocol describing simultaneous measurements of the ionic conductance through the nanotube lumen and across the nanotube wall, enabling spatiotemporal correlation between the nanotube constriction by Dyn1, leading to fission and membrane leakage. We present examples of "leaky" and "tight" fission reactions, specify the resolution limits of our method, and discuss how our results support the hemi-fission conjecture.

Mammalian PITPs at the Golgi and ER-Golgi Membrane Contact Sites

Phosphatidylinositol (PI)-transfer proteins (PITPs) have been long recognized as proteins that modulate phosphoinositide levels in membranes through their intrinsic PI/PC-exchange activity. Recent studies from flies and mammals suggest that certain PITPs bind phosphatidic acid (PA) and possess PI/PA-exchange activity. Phosphoinositides and PA play critical roles in cell signaling and membrane trafficking, and numerous biochemical, genetic and functional studies have shown that PITPs regulate cellular lipid metabolism, various signaling pathways and intracellular membrane transport events. In this mini-review, we discuss the function of mammalian PITPs at the Golgi and ER-Golgi membrane contact sites (MCS) and highlight DAG (Diacylglycerol) as a central hub of PITPs functions. We describe PITPs-associated phospho-signaling network at the ER-Golgi interface, and share our perspective on future studies related to PITPs at MCSs.

The Structure and Function of Acylglycerophosphate Acyltransferase 4/ Lysophosphatidic Acid Acyltransferase Delta (AGPAT4/LPAATδ).

Lipid-modifying enzymes serve crucial roles in cellular processes such as signal transduction (producing lipid-derived second messengers), intracellular membrane transport (facilitating membrane remodeling needed for membrane fusion/fission), and protein clustering (organizing lipid domains as anchoring platforms). The lipid products crucial in these processes can derive from different metabolic pathways, thus it is essential to know the localization, substrate specificity, deriving products (and their function) of all lipid-modifying enzymes. Here we discuss an emerging family of these enzymes, the lysophosphatidic acid acyltransferases (LPAATs), also known as acylglycerophosphate acyltransferases (AGPATs), that produce phosphatidic acid (PA) having as substrates lysophosphatidic acid (LPA) and acyl-CoA. Eleven LPAAT/AGPAT enzymes have been identified in mice and humans based on sequence homologies, and their localization, specific substrates and functions explored. We focus on one member of the family, LPAATδ, a protein expressed mainly in brain and in muscle (though to a lesser extent in other tissues); while at the cellular level it is localized at the trans-Golgi network membranes and at the mitochondrial outer membranes. LPAATδ is a physiologically essential enzyme since mice knocked-out for Lpaatδ show severe dysfunctions including cognitive impairment, impaired force contractility and altered white adipose tissue. The LPAATδ physiological roles are related to the formation of its product PA. PA is a multifunctional lipid involved in cell signaling as well as in membrane remodeling. In particular, the LPAATδ-catalyzed conversion of LPA (inverted-cone-shaped lipid) to PA (cone-shaped lipid) is considered a mechanism of deformation of the bilayer that favors membrane fission. Indeed, LPAATδ is an essential component of the fission-inducing machinery driven by the protein BARS. In this process, a protein-tripartite complex (BARS/14-3-3γ/phosphoinositide kinase PI4KIIIβ) is recruited at the trans-Golgi network, at the sites where membrane fission is to occur; there, LPAATδ directly interacts with BARS and is activated by BARS. The resulting formation of PA is essential for membrane fission occurring at those spots. Also in mitochondria PA formation has been related to fusion/fission events. Since PA is formed by various enzymatic pathways in different cell compartments, the BARS-LPAATδ interaction indicates the relevance of lipid-modifying enzymes acting exactly where their products are needed (i.e., PA at the Golgi membranes).

Rab‐mediated trafficking in the secondary cells of Drosophila male accessory glands and its role in fecundity

The male seminal fluid contains factors that affect female post‐mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only ~4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post‐mating response. To better understand the function of the secondary cells, we investigated their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole‐like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these organelles require Rab6 for their formation and many are essential in the process of creating the long‐term postmating behavior of females. In order to better serve the intracellular membrane and protein trafficking communities, we have created a searchable, online, open‐access imaging resource to display our complete findings regarding Rab localization in the accessory gland.

Modeling, dynamics and phosphoinositide binding of the pleckstrin homology domain of two novel PLCs: η1 and η2

PH domains mediate interactions involved in cell signaling, intracellular membrane transport regulation and cytoskeleton organization. Some PH domains bind phosphoinositides with different affinity and specificity. The two novel PLCη (1 and 2) possess an N-terminal PH domain (PHη1 and PHη2 respectively) that has been implicated in membrane association and induction of PLC activity. Understanding of the structure and dynamics is crucial for future modulation of lipid-protein interactions in PHη1, PHη2 and other PH domains. Therefore, the three-dimensional structure of PHη1 and PHη2 was modeled using ITASSER and phosphoinositides (IP3 and IP4) were docked in the inferred binding site using HADDOCK server. Molecular Dynamics simulations of unliganded and phosphoinositide bound PHη1 and PHη2 were performed using AMBER14 to study the mechanism of interaction, and conformational dynamics in response to phosphoinositide binding. The binding affinity was predicted using Kdeep server. The models of PHη1 and PHη2 had a conserved structural core consisting of seven β-strands and a C-terminal α-helix as seen in other PH domains. Sequence/structure analysis showed that phosphoinositide ligands bind PHη1 and PHη2 at the canonical binding site. Phosphoinositide binding induced movement of positively charged side chains towards the ligand, changes in the secondary structure especially at the β5-β6 loop and allosteric changes at the interface of β1-β2 and β5-β6 loops. Dynamics studies showed that the size of the binding site and differential affinity for IP3/IP4 binding is coordinated by the number, length, flexibility, secondary structure and allosteric interactions of the loops surrounding the phosphoinositide binding site.

Induction of Atypical Autophagy by Porcine Hemagglutinating Encephalomyelitis Virus Contributes to Viral Replication

Autophagy is a basic biological metabolic process involving in intracellular membrane transport pathways that recycle cellular components and eliminate intracellular microorganisms within the lysosome. Autophagy also plays an important part in virus infection and propagation. However, some pathogens, including viruses, have evolved unique trick to escape or exploit autophagy. This study explores the mechanism of autophagy induction by porcine hemagglutinating encephalomyelitis virus (PHEV) in Neuro-2a cells, and examines the role of autophagy in PHEV replication. PHEV triggered autophagy in Neuro-2a cells is dependent on the presence of bulk double- or single-membrane vacuoles, the accumulation of GFP-LC3 fluorescent dots, and the LC3 lipidation. In addition, PHEV induced an incomplete autophagic effect because the degradation level of p62 did not change in PHEV-infected cells. Further validation was captured using LysoTracker and lysosome-associated membrane protein by indirect immunofluorescence labeling in PHEV-infected cells. We also investigated the change in viral replication by pharmacological experiments with the autophagy inducer rapamycin or the autophagy inhibitor 3-MA, and the lysosomal inhibitor chloroquine (CQ). Suppression of autophagy by 3-MA increased viral replication, compared with the mock treatment, while promoting of autophagy by rapamycin reduced PHEV replication. CQ treatment enhanced the LC3 lipidation in PHEV-infected Neuro-2a cells but lowered PHEV replication. These results show that PHEV infection induces atypical autophagy and causes the appearance of autophagosomes but blocks the fusion with lysosomes, which is necessary for the replication of PHEV in nerve cells.