Distinct Subcellular Localization Research Articles

Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.

Systematic analysis of the lysine malonylome in Sanghuangporus sanghuang

BackgroundSanghuangporus sanghuang is a well-known traditional medicinal mushroom associated with mulberry. Despite the properties of this mushroom being known for many years, the regulatory mechanisms of bioactive compound biosynthesis in this medicinal mushroom are still unclear. Lysine malonylation is a posttranslational modification that has many critical functions in various aspects of cell metabolism. However, at present we do not know its role in S. sanghuang. In this study, a global investigation of the lysine malonylome in S. sanghuang was therefore carried out.ResultsIn total, 714 malonyl modification sites were matched to 255 different proteins. The analysis indicated that malonyl modifications were involved in a wide range of cellular functions and displayed a distinct subcellular localization. Bioinformatics analysis indicated that malonylated proteins were engaged in different metabolic pathways, including glyoxylate and dicarboxylate metabolism, glycolysis/gluconeogenesis, and the tricarboxylic acid (TCA) cycle. Notably, a total of 26 enzymes related to triterpene and polysaccharide biosynthesis were found to be malonylated, indicating an indispensable role of lysine malonylation in bioactive compound biosynthesis in S. sanghuang.ConclusionsThese findings suggest that malonylation is associated with many metabolic pathways, particularly the metabolism of the bioactive compounds triterpene and polysaccharide. This paper represents the first comprehensive survey of malonylation in S. sanghuang and provides important data for further study on the physiological function of lysine malonylation in S. sanghuang and other medicinal mushrooms.

Quantitative Subcellular Analysis of Cyclic Cell-Penetrating Peptide EJP18 in Nonadherent Cells.

Cyclization of cell-penetrating peptides (CPPs) often results in improved capacity for intracellular delivery of a range of cargoes but quantitating the distinct subcellular localization of them, and their linear counterparts, remains a challenge. Here we describe an optimized method for recombinant generation and purification of eGFP attached to the cyclic form of the newly discovered CPP EJP18 in E. coli. We also demonstrate a novel microscopy method for quantifying its subcellular distribution in leukemia cells.

Optical Control of Phosphoinositide Binding: Rapid Activation of Subcellular Protein Translocation and Cell Signaling.

Cells utilize protein translocation to specific compartments for spatial and temporal regulation of protein activity, in particular in the context of signaling processes. Protein recognition and binding to various subcellular membranes is mediated by a network of phosphatidylinositol phosphate (PIP) species bearing one or multiple phosphate moieties on the polar inositol head. Here, we report a new, highly efficient method for optical control of protein localization through the site-specific incorporation of a photocaged amino acid for steric and electrostatic disruption of inositol phosphate recognition and binding. We demonstrate general applicability of the approach by photocaging two unrelated proteins, sorting nexin 3 (SNX3) and the pleckstrin homology (PH) domain of phospholipase C delta 1 (PLCδ1), with two distinct PIP binding domains and distinct subcellular localizations. We have established the applicability of this methodology through its application to Son of Sevenless 2 (SOS2), a signaling protein involved in the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. Upon fusing the photocaged plasma membrane-targeted construct PH-enhanced green fluorescent protein (EGFP), to the catalytic domain of SOS2, we demonstrated light-induced membrane localization of the construct resulting in fast and extensive activation of the ERK signaling pathway in NIH 3T3 cells. This approach can be readily extended to other proteins, with minimal protein engineering, and provides a method for acute optical control of protein translocation with rapid and complete activation.

A sticky situation: regulation and function of protein palmitoylation with a spotlight on the axon and axon initial segment.

In neurons, the axon and axon initial segment (AIS) are critical structures for action potential initiation and propagation. Their formation and function rely on tight compartmentalisation, a process where specific proteins are trafficked to and retained at distinct subcellular locations. One mechanism which regulates protein trafficking and association with lipid membranes is the modification of protein cysteine residues with the 16-carbon palmitic acid, known as S-acylation or palmitoylation. Palmitoylation, akin to phosphorylation, is reversible, with palmitate cycling being mediated by substrate-specific enzymes. Palmitoylation is well-known to be highly prevalent among neuronal proteins and is well studied in the context of the synapse. Comparatively, how palmitoylation regulates trafficking and clustering of axonal and AIS proteins remains less understood. This review provides an overview of the current understanding of the biochemical regulation of palmitoylation, its involvement in various neurological diseases, and the most up-to-date perspective on axonal palmitoylation. Through a palmitoylation analysis of the AIS proteome, we also report that an overwhelming proportion of AIS proteins are likely palmitoylated. Overall, our review and analysis confirm a central role for palmitoylation in the formation and function of the axon and AIS and provide a resource for further exploration of palmitoylation-dependent protein targeting to and function at the AIS.

WW, PH and C-Terminal Domains Cooperate to Direct the Subcellular Localizations of PLEKHA5, PLEKHA6 and PLEKHA7.

PLEKHA5, PLEKHA6, and PLEKHA7 (WW-PLEKHAs) are members of the PLEKHA family of proteins that interact with PDZD11 through their tandem WW domains. WW-PLEKHAs contribute to the trafficking and retention of transmembrane proteins, including nectins, Tspan33, and the copper pump ATP7A, at cell-cell junctions and lateral membranes. However, the structural basis for the distinct subcellular localizations of PLEKHA5, PLEKHA6, and PLEKHA7 is not clear. Here we expressed mutant and chimeric proteins of WW-PLEKHAs in cultured cells to clarify the role of their structural domains in their localization. We found that the WW-mediated interaction between PLEKHA5 and PDZD11 is required for their respective association with cytoplasmic microtubules. The PH domain of PLEKHA5 is required for its localization along the lateral plasma membrane and promotes the lateral localization of PLEKHA7 in a chimeric molecule. Although the PH domain of PLEKHA7 is not required for its localization at the adherens junctions (AJ), it promotes a AJ localization of chimeric proteins. The C-terminal region of PLEKHA6 and PLEKHA7 and the coiled-coil region of PLEKHA7 promote their localization at AJ of epithelial cells. These observations indicate that the localizations of WW-PLEKHAs at specific subcellular sites, where they recruit PDZD11, are the result of multiple cooperative protein-lipid and protein-protein interactions and provide a rational basis for the identification of additional proteins involved in trafficking and sorting of WW-PLEKHAs.

Genome-Wide Identification and Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Suaeda aralocaspica, an Annual Halophyte With Single-Cellular C4 Anatomy

Phosphoenolpyruvate carboxylase (PEPC) plays pivotal roles in the carbon fixation of photosynthesis and a variety of metabolic and stress pathways. Suaeda aralocaspica belongs to a single-cellular C4 species and carries out a photosynthetic pathway in an unusually elongated chlorenchyma cell, which is expected to have PEPCs with different characteristics. To identify the different isoforms of PEPC genes in S. aralocaspica and comparatively analyze their expression and regulation patterns as well as the biochemical and enzymatic properties in this study, we characterized a bacterial-type PEPC (BTPC; SaPEPC-4) in addition to the two plant-type PEPCs (PTPCs; SaPEPC-1 and SaPEPC-2) using a genome-wide identification. SaPEPC-4 presented a lower expression level in all test combinations with an unknown function; two SaPTPCs showed distinct subcellular localizations and different spatiotemporal expression patterns but positively responded to abiotic stresses. Compared to SaPEPC-2, the expression of SaPEPC-1 specifically in chlorenchyma cell tissues was much more active with the progression of development and under various stresses, particularly sensitive to light, implying the involvement of SaPEPC-1 in a C4 photosynthetic pathway. In contrast, SaPEPC-2 was more like a non-photosynthetic PEPC. The expression trends of two SaPTPCs in response to light, development, and abiotic stresses were also matched with the changes in PEPC activity in vivo (native) or in vitro (recombinant), and the biochemical properties of the two recombinant SaPTPCs were similar in response to various effectors while the catalytic efficiency, substrate affinity, and enzyme activity of SaPEPC-2 were higher than that of SaPEPC-1 in vitro. All the different properties between these two SaPTPCs might be involved in transcriptional (e.g., specific cis-elements), posttranscriptional [e.g., 5′-untranslated region (5′-UTR) secondary structure], or translational (e.g., PEPC phosphorylation/dephosphorylation) regulatory events. The comparative studies on the different isoforms of the PEPC gene family in S. aralocaspica may help to decipher their exact role in C4 photosynthesis, plant growth/development, and stress resistance.

A Novel Core Effector Vp1 Promotes Fungal Colonization and Virulence of Ustilago maydis

The biotrophic fungus Ustilago maydis secretes a plethora of uncharacterized effector proteins and causes smut disease in maize. Among the effector genes that are up-regulated during the biotrophic growth in maize, we identified vp1 (virulence promoting 1), which has an expression that was up-regulated and maintained at a high level throughout the life cycle of the fungus. We characterized Vp1 by applying in silico analysis, reverse genetics, phenotypic assessment, microscopy, and protein localization and provided a fundamental understanding of the Vp1 protein in U. maydis. The reduction in fungal virulence and colonization in the vp1 mutant suggests the virulence-promoting function of Vp1. The deletion studies on the NLS (nuclear localization signal) sequence and the protein localization study revealed that the C-terminus of Vp1 is processed after secretion in plant apoplast and could localize to the plant nucleus. The Ustilago hordei ortholog UhVp1 lacks NLS localized in the plant cytoplasm, suggesting that the orthologs might have a distinct subcellular localization. Further complementation studies of the Vp1 orthologs in related smut fungi revealed that none of them could complement the virulence function of U. maydis Vp1, suggesting that UmVp1 could acquire a specialized function via sequence divergence.

Differential Expression of Drosophila Transgelins Throughout Development.

Transgelins are a conserved family of actin-binding proteins involved in cytoskeletal remodeling, cell contractility, and cell shape. In both mammals and Drosophila, three genes encode transgelin proteins. Transgelins exhibit a broad and overlapping expression pattern, which has obscured the precise identification of their role in development. Here, we report the first systematic developmental analysis of all Drosophila transgelin proteins, namely, Mp20, CG5023, and Chd64 in the living organism. Drosophila transgelins display overall higher sequence identity with mammalian TAGLN-3 and TAGLN-2 than with TAGLN. Detailed examination in different developmental stages revealed that Mp20 and CG5023 are predominantly expressed in mesodermal tissues with the onset of myogenesis and accumulate in the cytoplasm of all somatic muscles and heart in the late embryo. Notably, at postembryonic developmental stages, Mp20 and CG5023 are detected in the gut’s circumferential muscles with distinct subcellular localization: Z-lines for Mp20 and sarcomere and nucleus for CG5023. Only CG5023 is strongly detected in the adult fly in the abdominal, leg, and synchronous thoracic muscles. Chd64 protein is primarily expressed in endodermal and ectodermal tissues and has a dual subcellular localization in the cytoplasm and the nucleus. During the larval–pupae transition, Chd64 is expressed in the brain, eye, legs, halteres, and wings. In contrast, in the adult fly, Chd64 is expressed in epithelia, including the alimentary tract and genitalia. Based on the non-overlapping tissue expression, we predict that Mp20 and CG5023 mostly cooperate to modulate muscle function, whereas Chd64 has distinct roles in epithelial, neuronal, and endodermal tissues.

Complex functionality of protein phosphatase 1 isoforms in the heart

Protein phosphatase 1(PP1) is a key regulator of cardiac function through dephosphorylating serine/threonine residues within target proteins to oppose the function of protein kinases. Studies from failing hearts of animal models and human patients have demonstrated significant increase of PP1 activity in myocardium, while elevated PP1 activity in transgenic mice leads to cardiac dysfunction, suggesting that PP1 might be a therapeutic target to ameliorate cardiac dysfunction in failing hearts. In fact, cardiac overexpression of inhibitor 1, the endogenous inhibitor of PP1, increases cardiac contractility and suppresses heart failure progression. However, this notion of PP1 inhibition for heart failure treatment has been challenged by recent studies on the isoform-specific roles of PP1 in the heart. PP1 is a holoenzyme composed of catalytic subunits (PP1α, PP1β, or PP1γ) and regulatory proteins that target them to distinct subcellular locations for functional specificity. This review will summarize how PP1 regulates phosphorylation of some of the key cardiac proteins involved in Ca2+ handling and cardiac contraction, and the potential role of PP1 isoforms in controlling cardiac physiology and pathophysiology.

DeepSIRT: A deep neural network for identification of sirtuin targets and their subcellular localizations

Sirtuins are a family of proteins that play a key role in regulating a wide range of cellular processes including DNA regulation, metabolism, aging/longevity, cell survival, apoptosis, and stress resistance. Sirtuins are protein deacetylases and include in the class III family of histone deacetylase enzymes (HDACs). The class III HDACs contains seven members of the sirtuin family from SIRT1 to SIRT7. The seven members of the sirtuin family have various substrates and are present in nearly all subcellular localizations including the nucleus, cytoplasm, and mitochondria. In this study, a deep neural network approach using one-dimensional Convolutional Neural Networks (CNN) was proposed to build a prediction model that can accurately identify the outcome of the sirtuin protein by targeting their subcellular localizations. Therefore, the function and localization of sirtuin targets were analyzed and annotated to compartmentalize into distinct subcellular localizations. We further reduced the sequence similarity between protein sequences and three feature extraction methods were applied in datasets. Finally, the proposed method has been tested and compared with various machine-learning algorithms. The proposed method is validated on two independent datasets and showed an average of up to 85.77 % sensitivity, 97.32 % specificity, and 0.82 MCC for seven members of the sirtuin family of proteins.

The dystrophia myotonica WD repeat-containing protein DMWD and WDR20 differentially regulate USP12 deubiquitinase.

Despite its potential clinical relevance, the product of the DMWD (dystrophia myotonica, WD repeat containing) gene is a largely uncharacterized protein. The DMWD amino acid sequence is similar to that of WDR20, a known regulator of the USP12 and USP46 deubiquitinases (DUBs). Here, we apply a combination of insilico and experimental methods to investigate several aspects of DMWD biology. Molecular evolution and phylogenetic analyses reveal that WDR20 and DMWD, similar to USP12 and USP46, arose by duplication of a common ancestor during the whole genome duplication event in the vertebrate ancestor lineage. The analysis of public human gene expression datasets suggests that DMWD expression is positively correlated with USP12 expression in normal tissues and negatively correlated with WDR20 expression in tumors. Strikingly, a survey of the annotated interactome for DMWD and WDR20 reveals a largely nonoverlapping set of interactors for these proteins. Experimentally, we first confirmed that DMWD binds both USP12 and USP46 through direct coimmunoprecipitation of epitope-tagged proteins. We found that DMWD and WDR20 share the same binding interface in USP12, suggesting that their interaction with the DUB may be mutually exclusive. Finally, we show that both DMWD and WDR20 promote USP12 enzymatic activity, but they differentially modulate the subcellular localization of the DUB. Altogether, our findings suggest a model whereby mutually exclusive binding of DMWD and WDR20 to USP12 may lead to formation of deubiquitinase complexes with distinct subcellular localization, potentially targeting different substrate repertoires.

The canonical α-SNAP is essential for gametophytic development in Arabidopsis.

The development of male and female gametophytes is a pre-requisite for successful reproduction of angiosperms. Factors mediating vesicular trafficking are among the key regulators controlling gametophytic development. Fusion between vesicles and target membranes requires the assembly of a fusogenic soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) complex, whose disassembly in turn ensures the recycle of individual SNARE components. The disassembly of post-fusion SNARE complexes is controlled by the AAA+ ATPase N-ethylmaleimide-sensitive factor (Sec18/NSF) and soluble NSF attachment protein (Sec17/α-SNAP) in yeast and metazoans. Although non-canonical α-SNAPs have been functionally characterized in soybeans, the biological function of canonical α-SNAPs has yet to be demonstrated in plants. We report here that the canonical α-SNAP in Arabidopsis is essential for male and female gametophytic development. Functional loss of the canonical α-SNAP in Arabidopsis results in gametophytic lethality by arresting the first mitosis during gametogenesis. We further show that Arabidopsis α-SNAP encodes two isoforms due to alternative splicing. Both isoforms interact with the Arabidopsis homolog of NSF whereas have distinct subcellular localizations. The presence of similar alternative splicing of human α-SNAP indicates that functional distinction of two α-SNAP isoforms is evolutionarily conserved.

Loss of sphingosine kinase 2 enhances Wilm's tumor suppressor gene 1 and nephrin expression in podocytes and protects from streptozotocin-induced podocytopathy and albuminuria in mice

The sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is now appreciated as key regulatory factor for various cellular functions in the kidney, including matrix remodeling. It is generated by two sphingosine kinases (Sphk), Sphk1 and Sphk2, which are ubiquitously expressed, but have distinct enzymatic activities and subcellular localizations. In this study, we have investigated the role of Sphk2 in podocyte function and its contribution to diabetic nephropathy. We show that streptozotocin (STZ)-induced nephropathy and albuminuria in mice is prevented by genetic depletion of Sphk2. This protection correlated with an increased protein expression of the transcription factor Wilm's tumor suppressor gene 1 (WT1) and its target gene nephrin, and a reduced macrophage infiltration in immunohistochemical renal sections of STZ-treated Sphk2-/- mice compared to STZ-treated wildtype mice.To investigate changes on the cellular level, we used an immortalized human podocyte cell line and generated a stable knockdown of Sphk2 (Sphk2-kd) by a lentiviral transduction method. These Sphk2-kd cells accumulated sphingosine as a consequence of the knockdown, and showed enhanced nephrin and WT1 mRNA and protein expressions similar to the finding in Sphk2 knockout mice. Treatment of wildtype podocytes with the highly selective Sphk2 inhibitor SLM6031434 caused a similar upregulation of nephrin and WT1 expression. Furthermore, exposing cells to the profibrotic mediator transforming growth factor β (TGFβ) resulted on the one side in reduced nephrin and WT1 expression, but on the other side, in upregulation of various profibrotic marker proteins, including connective tissue growth factor (CTGF), fibronectin (FN) and plasminogen activator inhibitor (PAI) 1. All these effects were reverted by Sphk2-kd and SLM6031434. Mechanistically, the protection by Sphk2-kd may depend on accumulated sphingosine and inhibited PKC activity, since treatment of cells with exogenous sphingosine not only reduced the phosphorylation pattern of PKC substrates, but also increased WT1 protein expression. Moreover, the selective stable knockdown of PKCδ increased WT1 expression, suggesting the involvement of this PKC isoenzyme in WT1 regulation.The glucocorticoid dexamethasone, which is a treatment option in many glomerular diseases and is known to mediate a nephroprotection, not only downregulated Sphk2 and enhanced cellular sphingosine, but also enhanced WT1 and nephrin expressions, thus, suggesting that parts of the nephroprotective effect of dexamethasone is mediated by Sphk2 downregulation.Altogether, our data demonstrated that loss of Sphk2 is protective in diabetes-induced podocytopathy and can prevent proteinuria, which is a hallmark of many glomerular diseases. Thus, Sphk2 could serve as a new attractive pharmacological target to treat proteinuric kidney diseases.

PTENε suppresses tumor metastasis through regulation of filopodia formation

PTEN is one of the most frequently mutated genes in malignancies and acts as a powerful tumor suppressor. Tumorigenesis is involved in multiple and complex processes including initiation, invasion, and metastasis. The complexity of PTEN function is partially attributed to PTEN family members such as PTENα and PTENβ. Here, we report the identification of PTENε (also named as PTEN5), a novel N‐terminal‐extended PTEN isoform that suppresses tumor invasion and metastasis. We show that the translation of PTENε/PTEN5 is initiated from the CUG816 codon within the 5′UTR region of PTEN mRNA. PTENε/PTEN5 mainly localizes in the cell membrane and physically associates with and dephosphorylates VASP and ACTR2, which govern filopodia formation and cell motility. We found that endogenous depletion of PTENε/PTEN5 promotes filopodia formation and enhances the metastasis capacity of tumor cells. Overall, we identify a new isoform of PTEN with distinct subcellular localization and molecular function compared to the known members of the PTEN family. These findings advance our current understanding of the importance and diversity of PTEN functions.

Functional Role of Long-Chain Acyl-CoA Synthetases in Plant Development and Stress Responses.

Fatty acids (FAs) play vital roles in plants as components of lipid membranes that demarcate cells and organelles, as sources of stored energy in the form of neutral lipids, and as signaling molecules that elicit plant responses to adverse conditions. The activation of FAs through the formation of acyl-CoA intermediates by acyl-CoA synthetase (ACS) family enzymes is required for their synthesis and degradation. Long-chain ACSs (LACSs) represent a small subgroup of ACS enzymes that specifically convert long-chain or very-long-chain FAs into corresponding thioesters for multiple lipid-associated processes. Alteration of LACS activity often results in pleiotropic phenotypes such as male sterility, organ fusion, aberrant cuticular structure, delayed seed germination, altered seed oil content, and plant capacity to respond to various environmental stresses. This review provides a comprehensive analysis of LACS family enzymes including substrate specificity, tissue-specific expression patterns, and distinct subcellular localization highlighting their specific roles in lipid synthesis and degradation, the effects of altered LACS activity on plant development, the relationship between LACS activity and stress resistance, and the regulation of LACS activity. Finally, we pose several major questions to be addressed, which would advance our current understanding of LACS function in plants.

New Avenues for Phosphodiesterase Inhibitors in Asthma.

IntroductionPhosphodiesterases (PDEs) are isoenzymes ubiquitously expressed in the lungs where they catalyse cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (GMP), which are fundamental second messengers in asthma, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signaling pathways and, consequently, myriad biological responses. The superfamily of PDEs is composed of 11 families with a distinct substrate specificity, molecular structure and subcellular localization. Experimental studies indicate a possible role in asthma mainly for PDE3, PDE4, PDE5 and PDE7. Consequently, drugs that inhibit PDEs may offer novel therapeutic options for the treatment of this disease.Areas CoveredIn this article, we describe the progress made in recent years regarding the possibility of using PDE inhibitors in the treatment of asthma.Expert OpinionMany data indicate the potential benefits of PDE inhibitors as an add-on treatment especially in severe asthma due to their bronchodilator and/or anti-inflammatory activity, but no compound has yet reached the market as asthma treatment mainly because of their limited tolerability. Therefore, there is a growing interest in developing new PDE inhibitors with an improved safety profile. In particular, the research is focused on the development of drugs capable of interacting simultaneously with different PDEs, or to be administered by inhalation. CHF 6001 and RPL554 are the only molecules that currently are under clinical development but there are several new agents with interesting pharmacological profiles. It will be stimulating to assess the impact of such agents on individual treatable traits in specially designed studies.

Cellular expression and subcellular localization of diacylglycerol kinase γ in rat brain.

Gq protein-coupled receptors lead to activation of phospholipase C, which triggers phosphoinositide signaling. Diacylglycerol (DG) is one of the phosphoinositide metabolites and serves as a second messenger. Diacylglycerol kinase (DGK) phosphorylates DG to produce another second messenger phosphatidic acid. Of the DGK family, DGKγ is predominantly expressed in the brain at the mRNA level. Recent studies have shown the expression of DGKγ in vascular endothelial cells and adrenal medullary cells at the protein level, although its detailed cellular expression pattern and subcellular localization in the brain remain to be determined. In the present study, we addressed this point using specific DGKγ antibody. DGKγ was expressed in both projection neurons and interneurons in the cerebral cortex, hippocampal formation, and cerebellum. In cerebellar Purkinje cells, DGKγ was distributed to the soma and dendrites. Fractionation study revealed that DGKγ was enriched in the internal membranes containing the endoplasmic reticulum and Golgi complex. In immunoelectron microscopy, DGKγ was localized throughout the smooth endoplasmic reticulum system. These findings suggest that DGKγ shows unique cellular expression pattern in the brain and distinct subcellular localization different from other DGK isozymes.

Location, location, location: A compartmentalized view of TNF-induced necroptotic signaling.

Necroptosis is a lytic, proinflammatory cell death pathway, which has been implicated in host defense and, when dysregulated, the pathology of many human diseases. The central mediators of this pathway are the receptor-interacting serine/threonine protein kinases RIPK1 and RIPK3 and the terminal executioner, the pseudokinase mixed lineage kinase domain-like (MLKL). Here, we review the chronology of signaling along the RIPK1-RIPK3-MLKL axis and highlight how the subcellular compartmentalization of signaling events controls the initiation and execution of necroptosis. We propose that a network of modulators surrounds the necroptotic signaling core and that this network, rather than acting universally, tunes necroptosis in a context-, cell type-, and species-dependent manner. Such a high degree of mechanistic flexibility is likely an important property that helps necroptosis operate as a robust, emergency form of cell death.

Antigenicity and adhesiveness of a Plasmodium vivax VIR-E protein from Brazilian isolates.

BACKGROUND Plasmodium vivax, the major cause of malaria in Latin America, has a large subtelomeric multigene family called vir. In the P. vivax genome, about 20% of its sequences are vir genes. Vir antigens are grouped in subfamilies according to their sequence similarities and have been shown to have distinct roles and subcellular locations. However, little is known about vir subfamilies, especially when comes to their functions.OBJECTIVE To evaluate the diversity, antigenicity, and adhesiveness of Plasmodium vivax VIR-E.METHODS Vir-E genes were amplified from six P. vivax isolates from Manaus, North of Brazil. The presence of naturally acquired antibodies to recombinant PvBrVIR-E and PvAMA-1 was evaluated by ELISA. Binding capacity of recombinant PvBrVIR-E was assessed by adhesion assay to CHO-ICAM1 cells.FINDINGS Despite vir-E sequence diversity, among those identified sequences, a representative one was chosen to be expressed as recombinant protein. The presence of IgM or IgG antibodies to PvBrVIR-E was detected in 23.75% of the study population while the presence of IgG antibodies to PvAMA-1 antigen was 66.25% in the same population. PvBrVIR-E was adhesive to CHO-ICAM1.MAIN CONCLUSIONS PvBrVIR-E was antigenic and adhesive to CHO-ICAM1.