Plasma Membrane Localization Research Articles

Plasma Membrane Localization of CD36 Requires Vimentin Phosphorylation; A Mechanism by Which Macrophage Vimentin Promotes Atherosclerosis.

Vimentin is a type III intermediate filament protein expressed in cells of mesenchymal origin. Vimentin has been thought to function mainly as a structural protein and roles of vimentin in other cellular processes have not been extensively studied. Our current study aims to reveal functions of vimentin in macrophage foam cell formation, the critical stage of atherosclerosis. We demonstrated that vimentin null (Vim–/–) mouse peritoneal macrophages take up less oxidized LDL (oxLDL) than vimentin wild type (Vim+/+) macrophages. Despite less uptake of oxLDL in Vim–/– macrophages, Vim+/+ and Vim–/– macrophages did not show difference in expression of CD36 known to mediate oxLDL uptake. However, CD36 localized in plasma membrane was 50% less in Vim–/– macrophages than in Vim+/+ macrophages. OxLDL/CD36 interaction induced protein kinase A (PKA)-mediated vimentin (Ser72) phosphorylation. Cd36–/– macrophages did not exhibit vimentin phosphorylation (Ser72) in response to oxLDL. Experiments using phospho-mimetic mutation of vimentin revealed that macrophages with aspartate-substituted vimentin (V72D) showed more oxLDL uptake and membrane CD36. LDL receptor null (Ldlr–/–) mice reconstituted with Vim–/– bone marrow fed a western diet for 15 weeks showed 43% less atherosclerotic lesion formation than Ldlr–/– mice with Vim+/+ bone marrow. In addition, Apoe–/–Vim–/– (double null) mice fed a western diet for 15 weeks also showed 57% less atherosclerotic lesion formation than Apoe–/– and Vim+/+mice. We concluded that oxLDL via CD36 induces PKA-mediated phosphorylation of vimentin (Ser72) and phosphorylated vimentin (Ser72) directs CD36 trafficking to plasma membrane in macrophages. This study reveals a function of vimentin in CD36 trafficking and macrophage foam cell formation and may guide to establish a new strategy for the treatment of atherosclerosis.

Palmitoylation of the small GTPase Cdc42 by DHHC5 modulates spine formation and gene transcription

The small GTPase Cdc42 exists in the form of two alternatively spliced variants that are modified by hydrophobic chains: the ubiquitously expressed Cdc42-prenyl and a brain-specific isoform that can be palmitoylated, Cdc42-palm. Our previous work demonstrated that Cdc42-palm can be palmitoylated at two cysteine residues, Cys188 and Cys189, while Cys188 can also be prenylated. We showed that palmitoylation of Cys188 is essential for the plasma membrane localization of Cdc42-palm and is critically involved in Cdc42-mediated regulation of gene transcription and neuronal morphology. However, the abundance and regulation of this modification was not investigated. In the present study, we found that only a minor fraction of Cdc42 undergoes monopalmitoylation in neuroblastoma cells and in hippocampal neurons. In addition, we identified DHHC5 as one of the major palmitoyl acyltransferases that could physically interact with Cdc42-palm. We demonstrate that overexpression of dominant negative DHHC5 mutant decreased palmitoylation and plasma membrane localization of Cdc42-palm. In addition, knockdown of DHHC5 significantly reduced Cdc42-palm palmitoylation, leading to a decrease of Cdc42-mediated gene transcription and spine formation in hippocampal neurons. We also found that the expression of DHHC5 in the brain is developmentally regulated. Taken together, these findings suggest that DHHC5-mediated palmitoylation of Cdc42 represents an important mechanism for the regulation of Cdc42 functions in hippocampus.

Cryo-EM structure of human glucose transporter GLUT4

GLUT4 is the primary glucose transporter in adipose and skeletal muscle tissues. Its cellular trafficking is regulated by insulin signaling. Failed or reduced plasma membrane localization of GLUT4 is associated with diabetes. Here, we report the cryo-EM structures of human GLUT4 bound to a small molecule inhibitor cytochalasin B (CCB) at resolutions of 3.3 Å in both detergent micelles and lipid nanodiscs. CCB-bound GLUT4 exhibits an inward-open conformation. Despite the nearly identical conformation of the transmembrane domain to GLUT1, the cryo-EM structure reveals an extracellular glycosylation site and an intracellular helix that is invisible in the crystal structure of GLUT1. The structural study presented here lays the foundation for further mechanistic investigation of the modulation of GLUT4 trafficking. Our methods for cryo-EM analysis of GLUT4 will also facilitate structural determination of many other small size solute carriers.

Analysis of conditional colocalization relationships and hierarchies in three-color microscopy images.

Colocalization analysis of multicolor microscopy images is a cornerstone approach in cell biology. It provides information on the localization of molecules within subcellular compartments and allows the interrogation of known molecular interactions in their cellular context. However, almost all colocalization analyses are designed for two-color images, limiting the type of information that they reveal. Here, we describe an approach, termed "conditional colocalization analysis," for analyzing the colocalization relationships between three molecular entities in three-color microscopy images. Going beyond the question of whether colocalization is present or not, it addresses the question of whether the colocalization between two entities is influenced, positively or negatively, by their colocalization with a third entity. We benchmark the approach and showcase its application to investigate receptor-downstream adaptor colocalization relationships in the context of functionally relevant plasma membrane locations. The software for conditional colocalization analysis is available at https://github.com/kjaqaman/conditionalColoc.

Plasma membrane phosphatidylinositol (4,5)-bisphosphate is critical for determination of epithelial characteristics

Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.

A conserved asparagine residue in the inner surface of BRI1 superhelix is essential for protein native conformation

Asparagine-linked glycosylation (ALG, N-glycosylation) is one of the most prevalent protein modifications in eukaryotes and regulates protein folding, trafficking and function. Recently, we reported that the mutation of N154Q significantly led to the ER retention of brassinosteroids insensitive 1 (BRI1), the receptor of brassinosteroids (BRs). However, the mechanism of how the N154 site affects BRI1 structure is still not completely clear. In current study, we found that the removal of N154-glycan with S156A replacement significantly enhanced the ability of bri1 to complement bri1-301 mutant and plasma membrane localization compared with N154Q. In addition, the various mutations on N154 site resulted in bri1 retention in the ER, except for N154D. The 3D modeling suggested that there existed polar contacts around N154 site and the mutations not only destroyed the addition of N-glycan on the site, but also led to the disorder of hydrogen bonds formation. The sequence analysis showed that the N275 shared more similarity with N154 site and the removal of N275-glycan further enhanced the retention of bri1 carrying S156A mutation in the ER. Our results showed that N154 was special and essential for maintaining BRI1 structure and explored the role of those residues and key N-glycans lying in the LRR inner surface on protein conformation.

KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7.

Nonalcoholic steatohepatitis (NASH), characterized by hepatic steatosis, inflammation, and liver injury, has become a leading cause of end-stage liver diseases and liver transplantation. Krüppel-like factors 10 (KLF10) is a Cys2/His2 zinc finger transcription factor that regulates cell growth, apoptosis, and differentiation. However, whether it plays a role in the development and progression of NASH remains poorly understood. In the present study, we found that KLF10 expression was selectively upregulated in the mouse models and human patients with NASH, compared with simple steatosis (NAFL). Gain- and loss-of function studies demonstrated that hepatocyte-specific overexpression of KLF10 aggravated, whereas its depletion alleviated diet-induced NASH pathogenesis in mice. Mechanistically, transcriptomic analysis and subsequent functional experiments showed that KLF10 promotes hepatic lipid accumulation and inflammation through the palmitoylation and plasma membrane localization of fatty acid translocase CD36 via transcriptionally activation of zDHHC7. Indeed, both expression of zDHHC7 and palmitoylation of CD36 are required for the pathogenic roles of KLF10 in NASH development. Thus, our results identify an important role for KLF10 in NAFL-to-NASH progression through zDHHC7-mediated CD36 palmitoylation.

How Lipids Regulate the Cell Surface Localization of HSPA1A, a Stress‐inducible 70‐kDa Heat Shock Protein

HSPA1A is a molecular chaperone that regulates survival stressed and cancer cells. In addition to its cytosolic pro‐survival functions, HSPA1A also localizes at the plasma membrane (PM) of stressed and tumor cells. In cancer cells, the presence of HSPA1A at the cell surface allows the latter cells to develop resistance to radiation therapy, show increased invasiveness, and develop distant metastasis. Therefore, abolishing HSPA1A from the surface of tumor cells is a promising therapeutic. However, HSPA1A’s pathway to the cell surface remains enigmatic because this protein lacks membrane localization signals. Considering that HSPA1A binds with high selectivity to negatively charged lipids, like phosphatidylserine (PS) and mono‐phosphorylated phosphoinositides (PIPs), we hypothesized that the interaction of HSPA1A with these lipids allows the chaperone to translocate and anchor to the PM. To test this hypothesis, we subjected human cell lines to heat shock, depleted specific lipid targets, and quantified HSPA1A’s PM localization using confocal microscopy and cell surface biotinylation. These experiments revealed that depletion of PS, PI(4)P, and PI(3)P by co‐transfecting HSPA1A with known lipid‐biosensors, significantly reduced HSPA1A’s surface presentation. In contrast, reducing PI(4,5)P2 availability by overexpression of the PLCδ‐PH biosensor had minimal effects on HSPA1A’s PM‐localization. Next, we manipulated the cellular lipid content using ionomycin (a PLC activator), phenylarsine oxide (PAO) and GSK‐A1 (general and a selective PI4‐Kinase inhibitors, respectively), wortmannin (a PI3‐Kinase inhibitor), and fendiline (a repurposed FDA approved drug that reduces PS) using lipid‐biosensors as positive and negative controls. These experiments revealed that HSPA1A’s PM localization was unaffected by ionomycin but was greatly reduced in the presence of PAO, GSK‐A1, wortmannin, and fendiline, corroborating the findings obtained by the co‐transfection experiments. We further verified these results by selectively depleting PI(4,5)P2 and PI(4)P using a well‐established rapamycin‐induced phosphatase system. Our findings strongly support the notion that HSPA1A’s surface presentation is a multifaceted lipid‐driven phenomenon controlled by the binding of the chaperone to specific endosomal and plasma membrane lipids. These findings provide the basis for future interventions to render tumor cells sensitive to radiation therapy.

Insights into the role of N‐linked glycosylation in directing plasma membrane localization of GPCRs expressed in Saccharomyces cerevisiae

Glycosylation is the biochemical process of the attachment of sugar moieties or carbohydrates to proteins during co‐translational or post‐translational modification (PTM). It is one of many types of protein modifications that naturally occurs during the process of protein expression in organisms like Saccharomyces cerevisiae (Baker’s yeast). For natively expressed proteins, glycosylation can be critical for proper protein processing and can also be important for the production of proteins in a stable and biologically active state. For proteins that are non‐natively, or heterologously, expressed in these organisms, glycosylation can lead to improper processing and ultimately produce proteins with little to no biological relevance. This proves to be an undesirable effect for the elucidation of protein structure. However, large quantities of protein are required for structural analysis, presenting a significant obstacle for structural biologists and those who use heterologous expression as a means to acquire large quantities of protein. This problem becomes significantly more challenging for membrane proteins.Membrane proteins are an elusive class of biomolecules that reside or closely associate with the lipid bilayers of cells and encompass transporters, receptors and channel pores, to name a few. They play an intrinsic role in maintaining proper cell function including signaling and selective uptake of extracellular ions and small molecules. G‐protein coupled receptors (GPCRs) constitute one of the largest groups of membrane proteins across the plant and animal kingdoms. Despite being of scientific and medical interest, GPCRs and many integral membrane proteins are notoriously difficult to isolate and work with because of their hydrophobicity, structural flexibility and instability in the absence of the cell membrane. Optimized experimental conditions often vary from protein to protein and necessitate a systematic approach to achieve. This typically requires labor and resource intensive efforts. The goal of this work is to uncover new insights into the role of post‐translational processing and protein localization in the cell. Using a combination of fluorescence microscopy and bioinformatics we formulated an approach to investigate the effects of N‐linked glycosylation on plasma membrane localization of GPCRs expressed in S. cerevisiae. We postulate a critical link between plasma membrane localization and expression of “ligand active” or structurally relevant GPCRs. Previous studies of the human adenosine A2A receptor (hA2AR) show it readily localizes to the plasma membrane during expression in yeast, while the human dopamine D2 receptor (hD2R) does not. A sequence comparison of the two reveals three putative N‐linked glycosylation sites at the N‐terminal extracellular region of hD2R which is lacking in hA2AR. Using a chimera of these two receptors prepared with a C‐terminal GFP tag, we show the chimera does not traffic to the plasma membrane similarly to full‐length hA2AR, suggesting N‐linked glycosylation strongly influences functional high‐level expression of GPCRs and possibly other membrane proteins in yeast.

Secondary cell wall patterning-connecting the dots, pits and helices.

All plant cells are encased in primary cell walls that determine plant morphology, but also protect the cells against the environment. Certain cells also produce a secondary wall that supports mechanically demanding processes, such as maintaining plant body stature and water transport inside plants. Both these walls are primarily composed of polysaccharides that are arranged in certain patterns to support cell functions. A key requisite for patterned cell walls is the arrangement of cortical microtubules that may direct the delivery of wall polymers and/or cell wall producing enzymes to certain plasma membrane locations. Microtubules also steer the synthesis of cellulose-the load-bearing structure in cell walls-at the plasma membrane. The organization and behaviour of the microtubule array are thus of fundamental importance to cell wall patterns. These aspects are controlled by the coordinated effort of small GTPases that probably coordinate a Turing's reaction-diffusion mechanism to drive microtubule patterns. Here, we give an overview on how wall patterns form in the water-transporting xylem vessels of plants. We discuss systems that have been used to dissect mechanisms that underpin the xylem wall patterns, emphasizing the VND6 and VND7 inducible systems, and outline challenges that lay ahead in this field.

Cellular redox regulation by the neuronal glutamate and cysteine transporter, EAAT3

Glutamate transporters are essential for clearing glutamate from the extracellular space and thus limit the actions of glutamate within the brain. The neuronal glutamate transporter EAAT3 has also been shown to transport cysteine, which serves as a rate‐limiting substrate for the synthesis of neuronal glutathione, an essential regulator of oxidative stress in cells. Hence, EAAT3 can contribute to regulating cellular redox by acting as an essential cysteine transporter in neurons. EAAT3 mutations are also associated with several neuropsychiatric disorders such as schizophrenia and obsessive‐compulsive disorders.The activity of plasma membrane transporters can be altered by changes in trafficking, expression levels and recycling of the transporters. EAAT3 can rapidly cycle to and from the cell surface and through intracellular compartments within a few minutes and this can be regulated through several different intracellular signaling pathways. Previous studies have demonstrated that activation of protein kinase C (PKC) can regulate EAAT3 trafficking and increase the localization of EAAT3 at the membrane surface. There is evidence for some redox‐triggered mechanisms that can directly or indirectly influence signaling by altering the catalytic properties or influencing subcellular compartmentation of different PKC isoforms. This suggests a mechanism by which EAAT3 trafficking could be modulated by PKC as a response to cellular redox stress to increase cysteine import and facilitate the synthesis of the antioxidant glutathione. To study this, we monitored if oxidizing agents such as hydrogen peroxide can stimulate a redox response in the cell and lead to increased membrane localization of EAAT3. We found that application of hydrogen peroxide leads to an increase in EAAT3 membrane localization in Neuro2A cells expressing EAAT3. We used TIRF and biotinylation assays to confirm that EAAT3 localization can be altered by PKC activation and further examined whether this surface expression can be additionally modulated during redox stress.While studying downstream redox changes upon cysteine and antioxidant N‐acetyl cysteine treatments, we noticed a transient oxidative response in the mitochondria resulting from the generation of sulfane sulfur species and hydrogen sulfide in the mitochondria. To understand these downstream effects of cysteine and N‐acetyl cysteine on cytosolic and mitochondrial stress, we monitored the redox changes in the cytosol and mitochondria with live‐cell imaging using genetically encoded fluorescent redox sensors targeted to specific subcellular compartments. Using this approach, we were able to conduct live‐cell imaging with spatiotemporal accuracy and to dissect sub‐cellular changes in different redox couples caused by cysteine treatments. We also investigated cysteine transport and N‐acetyl cysteine transport through EAAT3, both of which appear crucial to cellular redox regulation in neuronal cells that have high mitochondrial activity and metabolic burden.

Identifying Vesicular Localization of the Rho GTPase RhoJ/TCL Through Colocalization with Rab Family GTPases

TCL belongs to the Rho family of GTPases and contributes to tumor‐associated angiogenesis. Understanding its basic cell biology is essential for establishing its role in this process. Experiments in several labs have shown plasma membrane and vesicular localizations of TCL, but it is less clear as to what subtype of vesicle the GTPase associates with and how it may influence vesicle trafficking. To better understand this aspect of TCL cellular biology, a YFP‐tagged version of TCL was co‐expressed in HeLa cells with sixteen different mCherry‐tagged versions of Rab GTPases. Images were captured using an Olympus Fluoview confocal microscope and colocalization assessed both qualitatively and quantitatively using CellSens software. CL colocalized with Rab5 and 7 as previously published but we also show colocalization with Rab17 and 40. Since Rab5, 7, and 17 Rab GTPases have reported vesicular localizations and functions, we intend to assess whether TCL plays any roles in their established pathways.

The Effects of Point Mutations on the Dimerization Domain of Ebola Virus Protein VP40

The Ebola Virus (EBOV) is a deadly virus first identified in 1976 from the Ebola River region of Zaire (presently the Democratic Republic of Congo). EBOV is known for causing viral hemorrhagic fever and it can cause a high mortality rate in infected patients. VP40 is the matrix protein of EBOV (eVP40), which has an N‐terminal domain responsible for dimerization and oligomerization of the protein to form filaments of dimers at the plasma membrane inner leaflet of the host cell. The eVP40 dimerization and filament formation is necessary for viral budding from the host cell membrane. This project aims to investigate which amino acid residues in this N‐terminal dimerization domain are necessary for the protein to be able to oligomerize at the plasma membrane inner leaflet. First, computational screens were performed to determine which residues may play a role in dimerization, and then mutant plasmids with changes at these residues harboring a green fluorescent protein tag were created. Based on the computational screen, we hypothesized amino acid residues Arg52, Ile54, Ala55, Asp56, Asp57, and His61 would be essential for dimerization of eVP40. Mutants of these residues were then transfected into Cos‐7 cells and imaged using confocal microscopy and compared to WT eVP40. Cellular images were analyzed using ImageJ to determine the amount of fluorescently‐tagged protein present at the plasma membrane. Confocal imaging confirmed our central hypothesis and the computational data as these mutations had a significant loss of eVP40 plasma membrane localization. The next steps in this project will be to perform virus‐like particle (VLP) collection to further analyze the effects of point mutations on the dimerization domain as it relates to VLP formation budding out of the host cell.

A Computational Method for the Visualization of Nitrated Hsp90 Distribution in 3D Culture Models

Protein tyrosine (Y) nitration is an oxidative post‐translational modification that occurs in pathological conditions but is absent in normal tissue. We previously showed that nitrated proteins support tumor cell survival and migration, and recently identified the chaperone Heat Shock Protein 90 (Hsp90) as the first protein that, when nitrated, acquires a novel tumorigenic activity in glioblastoma multiforme (GBM). While Hsp90 is expressed in all cells, we showed that nitration of Hsp90 on Y33 and/or Y56 induces a pathological gain‐of‐function. Nitration on Y33 (Hsp90NY33) decreases mitochondrial metabolism by inhibiting complex IV activity, while nitration on Y56 (Hsp90NY56) activates the P2X7 receptor (P2X7R), which is linked to increased glycolysis in tumors. Together, these metabolic changes are characteristics of the Warburg effect, a metabolic phenotype common amongst tumors, suggesting that nitrated Hsp90 may play a role as a metabolic switch driving tumor progression. Here, we developed a computational approach to visualize the subcellular and spatial distribution of differentially nitrated Hsp90 in 3D cell culture models of GBM (tumoroids) using confocal Z‐Stack images. Our automated, high‐throughput method takes Z‐stack outputs and generates a 3D heatmap of a differentially localized protein of interest, normalized to a housekeeping fluorescence signal. These normalized heatmaps can be generated from any fluorescence image and serve as an unbiased visualization tool to use in conjunction with fluorescence microscopy images. GBM tumoroids were fluorescently labelled for Hsp90NY33or Hsp90NY56using specific and selective in‐house monoclonal antibodies, and for mitochondrial complex IV and P2X7R. DAPI was used as nuclear stain and tumoroids were imaged using confocal laser scanning microscopy. We observed a ~50% loss in fluorescence signal across the depth of tumoroid Z‐stacks, a common problem related to tissue penetration of laser light that leads to an underrepresented fluorescent signal at the core and bottom of imaged tumoroids. To overcome this problem, Python was used to analyze each focal plane within a confocal Z‐stack, normalizing the signal corresponding to the protein of interest by the mean DNA content (DAPI signal) at each focal plane. This normalized signal was used to generate a 3D heatmap of the protein of interest within the tumoroids. Method‐generated heatmaps confirmed the intracellular localization of Hsp90NY33in mitochondria and cytosol, and a distinct localization in the tumoroid periphery. In contrast, Hsp90NY56 was also localized in the nucleus, and was homogeneously distributed throughout the tumoroid. P2X7R showed a clear cell membrane localization, with increased expression in the tumoroid periphery. This suggests that Hsp90NY33may reduce aerobic respiration in the periphery of the tumoroid, allowing oxygen diffusion into the core, while Hsp90NY56 supports glycolysis throughout the tumoroid. Our results provide a novel fluorescence image analysis tool with potential to be expanded for several other 2D and 3D imaging modalities.

Targeted autophagy disruption reveals the central role of macrophage iron metabolism in systemic iron homeostasis

AbstractIron homeostasis depends on both intracellular control through iron-responsive proteins and the systemic level of iron through hepcidin-ferroportin axis. Indeed, the hormone hepcidin downregulates the ferroportin iron exporter to control iron recycling from macrophages and iron uptake from enterocytes. Here, we focused on the role of autophagy in macrophage iron metabolism and systemic iron homeostasis. Mice deficient for autophagy in macrophages (LysM-Atg5−/−) mimicked a primary iron overload phenotype, resulting in high ferroportin expression in both macrophages and enterocytes that correlated with marked parenchymal iron overload. Furthermore, LysM-Atg5−/− mice exhibited increased hematopoietic activity with no sign of anemia but correlating with rather high plasma iron level. Compared with wild-type cells, bone marrow–derived macrophages from LysM-Atg5−/− mice had significantly increased ferroportin expression and decreased iron content, confirming high iron export. In erythrophagocytic macrophages, autophagy regulates hemosiderin storage mechanisms as well as degradation of ferroportin and subsequently its plasma membrane localization and iron export; furthermore, ferroportin colocalization with hepcidin indicates hepcidin autocrine activity. Relatively high hepatic hepcidin expression and decreased hepcidin level in the spleen of LysM-Atg5−/− mice, correlating with low hemosiderin iron storage, as well as in erythrophagocytic Atg5−/− macrophages were evidenced. Therefore, our results highlight the critical role of autophagy in macrophages for iron trafficking and systemic iron homeostasis. We propose that in macrophages, autophagy restricts ferroportin level and iron export, resulting in hepcidin expression with an autocrine-paracrine effect that plays a role in the regulation of ferroportin expression in duodenal enterocytes.

Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein

Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multisynthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that participates in tRNA trafficking; we show that tRip also functions as an AIMP. We identified three aaRSs, the glutamyl-tRNA synthetase (ERS), glutaminyl-tRNA synthetase (QRS), and methionyl-tRNA synthetase (MRS), which were specifically coimmunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal glutathione-S-transferase (GST)–like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip–ERS–QRS) and the M-complex (tRip–ERS–MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together, our results demonstrate that neither the singular homodimerization of tRip nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host–pathogen interaction is discussed.

The involvement of oxysterol-binding protein related protein (ORP) 6 in the counter-transport of phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) in neurons

Oxysterol-binding protein (OSBP)-related protein (ORP) 6, a member of subfamily III in the ORP family, localizes to membrane contact sites between the endoplasmic reticulum (ER) and other organelles and functions in non-vesicular exchange of lipids including phosphatidylinositol-4-phosphate (PI4P) in neurons. In this study, we searched for the lipid counter-transported in exchange for PI4P by using molecular cell biology techniques. Deconvolution microscopy revealed that knockdown of ORP6 partially shifted localization of a phosphatidylserine (PS) marker but not filipin in primary cultured cerebellar neurons. Overexpression of ORP6 constructs lacking the OSBP-related ligand binding domain (ORD) resulted in the same shift of the PS marker. A PI4KⅢα inhibitor specifically inhibiting the synthesis and plasma membrane (PM) localization of PI4P, suppressed the localization of ORP6 and the PS marker at the PM. Overexpression of mutant PS synthase 1 (PSS1) inhibited transport of the PS marker to the PM and relocated the PI4P marker to the PM in Neuro-2A cells. Introduction of ORP6 but not the dominant negative ORP6 constructs, shifted the localization of PS back to the PM. These data collectively suggest the involvement of ORP6 in the counter-transport of PI4P and PS.

Enteroaggregative Escherichia coli induces altered glycosylation in membrane proteins of cultured human intestinal epithelial cells

Emerging evidences have suggested that pathogens are capable of manipulating the glycosylation pattern of host-cell glycoconjugates, which may promote their attachment to these cells. Several enteric pathogens are known to induce such altered glycosylation in intestinal epithelium thereby, facilitating the disease process. Enteroaggregative Escherichia coli (EAEC), is one of such pathogens, known to cause acute and persistent diarrhea worldwide. However, glycosylation modulation due to EAEC infection has not been explored so far. In this study, EAEC-induced glycosylation changes in membrane proteins of human small-intestinal and colonic epithelial cell lines were found as revealed by lectin-overlay transblotting using four lectins, among which Sambucus nigra agglutinin (SNA) was selected for subsequent experiments. Several differentially expressed membrane-proteins were detected on SNA-overlay transblots following 2D-PAGE and identified by MALDI-TOF/TOF mass spectrometric analysis. Among these, voltage-dependent anion-selective channel-protein 2 (VDAC2) and prohibitin 2 (PHB2), common to both the cell lines were chosen for further characterization. Reactivity of these proteins to SNA was substantiated by their presence in SNA-agarose affinity chromatography eluted fractions. The plasma membrane localization of VDAC2 and PHB2 in EAEC infected cell lines was validated by confocal microscopy. These proteins were characterized as sialoglycoproteins by SNA-overlay transblots in presence a specific SNA inhibitor i.e., 6'sialyl lactose and deglycosylation using PNGase F, O-glycosidase and neuraminidase. Membrane localization of these sialoglycoproteins was found to facilitate EAEC adherence to human intestinal epithelial cells. SignificanceOur findings regarding EAEC induced altered glycosylation pattern of host cell membrane proteins may help in better understanding of the disease pathogenesis.

Targeting farnesylation as a novel therapeutic approach in HRAS-mutant rhabdomyosarcoma

Activating RAS mutations are found in a subset of fusion-negative rhabdomyosarcoma (RMS), and therapeutic strategies to directly target RAS in these tumors have been investigated, without clinical success to date. A potential strategy to inhibit oncogenic RAS activity is the disruption of RAS prenylation, an obligate step for RAS membrane localization and effector pathway signaling, through inhibition of farnesyltransferase (FTase). Of the major RAS family members, HRAS is uniquely dependent on FTase for prenylation, whereas NRAS and KRAS can utilize geranylgeranyl transferase as a bypass prenylation mechanism. Tumors driven by oncogenic HRAS may therefore be uniquely sensitive to FTase inhibition. To investigate the mutation-specific effects of FTase inhibition in RMS we utilized tipifarnib, a potent and selective FTase inhibitor, in in vitro and in vivo models of RMS genomically characterized for RAS mutation status. Tipifarnib reduced HRAS processing, and plasma membrane localization leading to decreased GTP-bound HRAS and decreased signaling through RAS effector pathways. In HRAS-mutant cell lines, tipifarnib reduced two-dimensional and three-dimensional cell growth, and in vivo treatment with tipifarnib resulted in tumor growth inhibition exclusively in HRAS-mutant RMS xenografts. Our data suggest that small molecule inhibition of FTase is active in HRAS-driven RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with RMS.

RIP3 Contributes to Cardiac Hypertrophy by Influencing MLKL-Mediated Calcium Influx.

Receptor-interacting protein 3(RIP3), a RIP family member, has been reported as a critical regulator of necroptosis and involves in the pathogenesis of various heart diseases. However, its role in the development of myocardial hypertrophy after pressure overload is unclear. We aimed to investigate the roles of RIP3 in pathological cardiac hypertrophy. A rat model of myocardial hypertrophy induced by the aortic banding method was used in this study. Neonatal rat cardiomyocytes (NRCMs) were stimulated with angiotensin II (Ang-II) or phenylephrine (PE) to induce neurohumoral stress. Our results showed that RIP3 level was significantly elevated in the hypertrophic myocardium tissues from patients, rats subjected to AB surgery, and NRCMs treated with Ang-II or PE. After downregulation of RIP3 expression in NRCMs, the phenotypes of myocardial hypertrophy were obviously alleviated. In mechanism, we demonstrated that RIP3 interacts with mixed lineage kinase domain-like protein (MLKL) and promotes its cell membrane localization to increase the influx of calcium within cells, thereby mediating the development of myocardial hypertrophy. More interestingly, we found the blockage of calcium influx by 2-aminoethoxydiphenyl borate, and lanthanum chloride efficiently reverses RIP3-induced cardiac remodeling in NRCMs. Taken together, our findings indicate a key role of the RIP3-MLKL signaling pathway in myocardial hypertrophy, which may be a novel promising treatment strategy for myocardial hypertrophy.