Cytoplasmic Face Research Articles

Bacterial β-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2).

Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 β-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.

Unlocking the Bacterial SecY Translocon

SummaryThe Sec translocon performs protein secretion and membrane protein insertion at the plasma membrane of bacteria and archaea (SecYEG/β), and the endoplasmic reticular membrane of eukaryotes (Sec61). Despite numerous structures of the complex, the mechanism underlying translocation of pre-proteins, driven by the ATPase SecA in bacteria, remains unresolved. Here we present a series of biochemical and computational analyses exploring the consequences of signal sequence binding to SecYEG. The data demonstrate that a signal sequence-induced movement of transmembrane helix 7 unlocks the translocon and that this conformational change is communicated to the cytoplasmic faces of SecY and SecE, involved in SecA binding. Our findings progress the current understanding of the dynamic action of the translocon during the translocation initiation process. The results suggest that the converging effects of the signal sequence and SecA at the cytoplasmic face of SecYEG are decisive for the intercalation and translocation of pre-protein through the SecY channel.

Regulation of O-antigen biosynthesis in Yersinia enterocolitica.

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane (OM) of Gram-negative bacteria. The LPS molecule consists of three regions: (i) the lipid A, a polar glycolipid in which a disaccharide backbone is replaced with six or seven fatty acid; (ii) the core, an oligosaccharide often rich in negatively charged groups and, (iii) the O-antigen, a polysaccharide that protrudes into the surroundings. It is widely known that the lipid A part is responsible for most of the biological effects of LPS; however, there is increasing body of evidence showing that O-antigen modulates the biological activity of lipid A and also plays a critical role in the initial interaction of bacterium with the host. The biosynthesis of heteropolymeric O-antigens whose O-units are composed of different sugar residues starts in the cytoplasm by the activation of sugar 1-phosphates in a reaction with one of the nucleoside triphosphates, followed by different biosynthetic pathways to give rise to cytoplasmically located individual nucleoside diphosphate (NDP)-activated sugar precursors. The assembly of the O-units takes place on the cytoplasmic face of the inner membrane and involves the transfer of sugar residues from the NDP-sugar precursors onto a lipid carrier molecule, undecaprenylphosphate (Und-P), by specific glycosyltransferases. The completed O-units still assembled on the Und-P are translocated by the O-unit flippase, Wzx, across the inner membrane to the periplasmic face. There the O-units are polymerized by the O-antigen polymerase, Wzy, into an O-antigen that is ligated to the lipid Acore structure by the O-antigen ligase encoded by the waaL gene of the core

The Trypanosome Nuclear Pore Reveals 1.5 Billion Years of Similarities and Differences.

The nuclear pore complex (NPC) is perhaps the most magnificent protein complex in the eukaryotic cell. It is built from almost 500 individual protein molecules of about 30 different types, arranged in 8-fold symmetry to create a central pore through which proteins, RNAs, and all other molecules must pass in order to enter or exit the nucleus. The nucleus and the nuclear membrane may define what it means to be a eukaryote, but it is the NPC that makes eukaryotic existence possible. Given its functional importance, you might think that the NPCs of all eukaryotes would be the same—that the constraints of natural selection might have prevented any variation from some successful early model. But in this issue of PLOS Biology, Samson Obado, Mark Field, Brian Chait, Michael Rout, and colleagues show that the NPC of the parasite Trypanosoma brucei differs in some important ways from those of vertebrates, plants, and yeast (Fig 1). Their findings shed light on the evolution of the NPC, strengthening the argument that it may have originated as a far simpler progenitor. Fig 1 Conservation and divergence at the nuclear rim. The choice to study T. brucei, which causes African sleeping sickness, was no random one—trypanosomes branched from the rest of the eukaryote lineage quite early. Vertebrates, plants, and yeast—whose NPCs have been characterized in detail—are more closely related to each other than any of them are to the trypanosomes. Thus, the new study provided an opportunity to compare the structural similarities and differences between groups that have been separated for over 1.5 billion years. The authors’ group had previously identified 22 T. brucei nucleoporins (or Nups, as all the proteins of the nuclear pore complex are known), which they identified as orthologs (related by descent and presumed function) of Nups from yeast or humans. Here, they performed “affinity capture/mass spectrometry interactomics” to better characterize the functional interactions of the T. brucei Nups and to discover new ones. The process of affinity capture uses a known protein as the bait to find the set of proteins to which it binds. Each Nup was tagged in succession with green fluorescent protein (GFP) (which, remarkably, did not interfere with the function of the pore complex) and was then purified and captured by an antibody to the GFP. Depending on the stringency of the purification process, the GFP-tagged Nup brought along with it one, some, or all of the other proteins to which it was bound, either directly or indirectly, in the various subunits of the NPC. By repeating this process with each known Nup, the authors were able to, as they put it, “walk out” from the new protein to characterize its neighborhood, leading to a detailed map of the interactions of all T. brucei Nups, as well as to the discovery and characterization of five new Nups. Electron microscopy of Nups labeled with electron-dense gold provided further details on the locations of the Nups within the complex as a whole. The most conserved portion of the T. brucei NPC was the inner ring, composed of a set of proteins that line the pore itself. The authors found that the overall architecture of the ring, and the positioning of individual Nups within it, matched that seen in vertebrates, yeast, and plants. Quite the opposite was true of the membrane-anchoring proteins:, no trypanosome orthologs were found of several Nups known from yeast and humans, and one of the new trypanosome Nups employed a transmembrane domain as part of its anchoring structure, which is not found in other eukaryotes. Perhaps the most surprising discovery concerned the distribution of the FG-Nups, a set of proteins rich in phenylalanine (F) and glycine (G) residue repeats, which project into the central pore and are thought to interact with the proteins that mediate cargo transport. In vertebrates, yeast, and plants, a quarter of FG-Nups are restricted to either the cytoplasmic or nucleoplasmic faces of the NPC, and that asymmetry helps drive export of messenger RNA. In T. brucei, the FG-Nups were largely symmetrically distributed, including the ortholog of a cytoplasmically restricted protein in yeast, implying a different mechanism of mRNA export. The confirmation that the structure of the inner core is highly conserved strengthens the model of NPC evolution previously proposed by the authors, in which the ancestral complex arose from multiple duplication events of a much simpler monomer that coated and stabilized openings in the nuclear membrane. The divergent membrane anchoring systems likely arose or were elaborated after the trypanosomes separated from the rest of the eukaryote pack, while the more symmetric distribution of their FG-Nups may reflect an ancestral condition, which the authors suggest was followed by development of asymmetry.

Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex.

The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes.

VAP, a Versatile Access Point for the Endoplasmic Reticulum: Review and analysis of FFAT-like motifs in the VAPome

Dysfunction of VAMP-associated protein (VAP) is associated with neurodegeneration, both Amyotrophic Lateral Sclerosis and Parkinson’s disease. Here we summarize what is known about the intracellular interactions of VAP in humans and model organisms. VAP is a simple, small and highly conserved protein on the cytoplasmic face of the endoplasmic reticulum (ER). It is the sole protein on that large organelle that acts as a receptor for cytoplasmic proteins. This may explain the extremely wide range of interacting partners of VAP, with components of many cellular pathways binding it to access the ER. Many proteins that bind VAP also target other intracellular membranes, so VAP is a component of multiple molecular bridges at membrane contact sites between the ER and other organelles. So far approximately 100 proteins have been identified in the VAP interactome (VAPome), of which a small minority have a “two phenylalanines in an acidic tract” (FFAT) motif as it was originally defined. We have analyzed the entire VAPome in humans and yeast using a simple algorithm that identifies many more FFAT-like motifs. We show that approximately 50% of the VAPome binds directly or indirectly via the VAP-FFAT interaction. We also review evidence on pathogenesis in genetic disorders of VAP, which appear to arise from reduced overall VAP levels, leading to ER stress. It is not possible to identify one single interaction that underlies disease. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Molecular Architecture of the Nup82 Complex, the Cytoplasmic mRNA Export Platform in the Nuclear Pore Complex

mRNA biogenesis is an essential and intricate process that occurs within the nucleus and ends with the mRNA export through the nuclear pore complex (NPC), a conserved process which defects lead to serious human diseases. The protein assemblies that perform the last steps in mRNA export are located at the cytoplasmic face of the Nuclear Pore complex (NPC). Here we describe the molecular architecture of the NPC cytoplasmic mRNA export machinery. Through an integrative modeling approach that combines various sources of data, including chemical cross-linking, electron microscopy, and small angle X-ray scattering, we generate a hybrid structure of the native Nup82 complex, the main component of the assembly. Correlative phenotypic mapping and structural data from chemical cross-linking reveal how the Nup82 complex docks into the Nup84 complex at the cytoplasmic face of the NPC and allows us to generate a first map of the associated mRNA remodeling machinery. Our map serves as a framework to understand the mRNA export process and NPC function.

GRASPs in Golgi Structure and Function.

The Golgi apparatus is a central intracellular membrane organelle for trafficking and modification of proteins and lipids. Its basic structure is a stack of tightly aligned flat cisternae. In mammalian cells, dozens of stacks are concentrated in the pericentriolar region and laterally connected to form a ribbon. Despite extensive research in the last decades, how this unique structure is formed and why its formation is important for proper Golgi functioning remain largely unknown. The Golgi ReAssembly Stacking Proteins, GRASP65, and GRASP55, are so far the only proteins shown to function in Golgi stacking. They are peripheral membrane proteins on the cytoplasmic face of the Golgi cisternae that form trans-oligomers through their N-terminal GRASP domain, and thereby function as the “glue” to stick adjacent cisternae together into a stack and to link Golgi stacks into a ribbon. Depletion of GRASPs in cells disrupts the Golgi structure and results in accelerated protein trafficking and defective glycosylation. In this minireview we summarize our current knowledge on how GRASPs function in Golgi structure formation and discuss why Golgi structure formation is important for its function.

High-Resolution Scanning Electron Microscopy and Immuno-Gold Labeling of the Nuclear Lamina and Nuclear Pore Complex.

Scanning electron microscopy (SEM) is a technique used to image surfaces. Field emission SEMs (feSEMs) can resolve structures that are ~0.5-1.5 nm apart. FeSEM, therefore is a useful technique for imaging molecular structures that exist at surfaces such as membranes. The nuclear envelope consists of four membrane surfaces, all of which may be accessible for imaging. Imaging of the cytoplasmic face of the outer membrane gives information about ribosomes and cytoskeletal attachments, as well as details of the cytoplasmic peripheral components of the nuclear pore complex, and is the most easily accessed surface. The nucleoplasmic face of the inner membrane is easily accessible in some cells, such as amphibian oocytes, giving valuable details about the organization of the nuclear lamina and how it interacts with the nuclear pore complexes. The luminal faces of both membranes are difficult to access, but may be exposed by various fracturing techniques. Protocols are presented here for the preparation, labeling, and feSEM imaging of Xenopus laevis oocyte nuclear envelopes.

Endosomal integrin signals for survival.

The mechanisms underlying integrin-dependent signalling are a topic of continued study. Endocytosed integrins are now shown to drive assembly of signalling complexes on the cytoplasmic face of endocytic membranes to promote cancer cell survival and increase metastatic capacity following cell detachment.

Grain setting defect1 (GSD1) function in rice depends on S-acylation and interacts with actin 1 (OsACT1) at its C-terminal.

Grain setting defect1 (GSD1), a plant-specific remorin protein specifically localized at the plasma membrane (PM) and plasmodesmata of phloem companion cells, affects grain setting in rice through regulating the transport of photoassimilates. Here, we show new evidence demonstrating that GSD1 is localized at the cytoplasmic face of the PM and a stretch of 45 amino acid residues at its C-terminal is required for its localization. Association with the PM is mediated by S-acylation of cysteine residues Cys-524 and Cys-527, in a sequence of 45 amino acid residues essential for GSD1 function in rice. Furthermore, the coiled-coil domain in GSD1 is necessary for sufficient interaction with OsACT1. Together, these results reveal that GSD1 attaches to the PM through S-acylation and interacts with OsACT1 through its coiled-coil domain structure to regulate plasmodesmata conductance for photoassimilate transport in rice.

The N-terminal domains determine cellular localization and functions of the Doa4 and Ubp5 deubiquitinating enzymes

Ubiquitination is involved in numerous cellular regulatory mechanisms including the cell cycle, signal transduction and quality control. Ubiquitin modifies proteins by consecutive actions of ubiquitin-activating/conjugating enzymes. Attachment of ubiquitin is reversible. Deubiquitinating enzymes are responsible for removal of ubiquitin from ubiquitin-protein conjugates. Genome of the yeast Saccharomyces cerevisiae encodes structurally related but functionally distinct enzymes − Doa4 and Ubp5. Doa4 is involved in general ubiquitin-dependent proteolysis and is responsible for deubiquitination of ubiquitin-protein conjugates at the cytoplasmic face of the late endosome. The N-terminal domain targets the enzyme to the endosome membrane after ESCRT-III complex has formed there. By contrast, corresponding region of homologous Ubp5 is critical for its bud neck localization in dividing cells. Conceivably, Ubp5 plays an essential role in cytokinesis. Here we show that Doa4 physically interacts with the ESCRT-III component Snf7 and preferentially cleaves Lys63-linked ubiquitin oligomers involved in membrane protein trafficking. We also demonstrate that the unstable regulator of cytokinesis Hof1 accumulates in proteasomal mutants and is required for cellular localization of Ubp5.

PIP2 and Talin Join Forces to Activate Integrin.

Integrins are major players in cell adhesion and migration, and malfunctions in controlling their activity are associated with various diseases. Nevertheless, the details of integrin activation are not completely understood, and the role of lipids in the process is largely unknown. Herein, we show using atomistic molecular dynamics simulations that the interplay of phosphatidylinositol 4,5-bisphosphate (PIP2) and talin may directly alter the conformation of integrin αIIbβ3. Our results provide a new perspective on the role of PIP2 in integrin activation and indicate that the charged PIP2 lipid headgroup can perturb a clasp at the cytoplasmic face of the integrin heterodimer.

Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p.

UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsid-membrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. Membrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to the function of UL36p in capsid envelopment.

Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUS-associated ALS.

Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER-Golgi transport is a novel therapeutic target in ALS.

Abstract 5007: A novel polyisoprenylated cysteinyl amide inhibitor, NSL-BA-055, selectively inhibits proliferation of hepatocellular carcinoma cells

Abstract The monomeric GTP-ase, Ras, is mutated in over 30% of cancers where it promotes uncontrolled proliferation of cancer cells. Ras signaling is one of many pathophysiological pathways implicated in hepatocellular carcinoma that results in cell survival, differentiation, proliferation, and angiogenesis. Ras is bound to the cytoplasmic face of the plasma membrane where it relays intracellular signals. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) effectively disrupt the functions of polyisoprenylated proteins such as Ras. Many hepatocellular carcinomas (HCCs) have dysregulated Ras signaling. However, the effects of PCAIs on hepatocellular carcinoma (HCC) are unknown. The aim of this study was to investigate the effect of a novel PCAI, NSL-BA-055, on HCC. Concentrations of NSL-BA-055 ranging from 0 to 10 μM were used for experimentation. Effects on proliferation of BNL CL.2 (non-tumorigenic mouse hepatocytes) and BNL 1ME A.7R.1 (mouse HCC cells derived from BNL CL.2 hepatocytes) were analyzed. Next, the effects of NSL-BA-055 on Akt protein expression, a downstream component of Ras signaling, were examined by western blotting. NSL-BA-055 significantly inhibited the proliferation of BNL 1ME A. 7R.1 HCC cells, but not the non-tumorigenic BNL CL.2 hepatocytes. Also, concentrations of the NSL-BA-055 compound as low as 4 μM significantly inhibited the proliferation of BNL 1ME A.7R.1 HCC cells. Western blotting revealed that NSL-BA-055 inhibited Akt protein expression at 4μM. Therefore PCAIs such as NSL-BA-055 is a novel class of agents that selectively inhibit the proliferation of HCC cells, but not normal hepatocytes. This effect may be via inhibition of Akt, a downstream intermediate of Ras signaling. Consequently, NSL-BA-055 is a novel candidate agent for HCC therapy. Note: This abstract was not presented at the meeting. Citation Format: Michelle K. Naidoo, Nazarius Lamango, Olorunseun O. Ogunwobi. A novel polyisoprenylated cysteinyl amide inhibitor, NSL-BA-055, selectively inhibits proliferation of hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2015-5007

N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin.

The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER) in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases.

SiRNA Screening Identifies the Host Hexokinase 2 (HK2) Gene as an Important Hypoxia-Inducible Transcription Factor 1 (HIF-1) Target Gene in Toxoplasma gondii-Infected Cells.

ABSTRACTAlthough it is established that oxygen availability regulates cellular metabolism and growth, little is known regarding how intracellular pathogens use host factors to grow at physiological oxygen levels. Therefore, large-scale human small interfering RNA screening was performed to identify host genes important for growth of the intracellular protozoan parasite Toxoplasma gondii at tissue oxygen tensions. Among the genes identified by this screen, we focused on the hexokinase 2 (HK2) gene because its expression is regulated by hypoxia-inducible transcription factor 1 (HIF-1), which is important for Toxoplasma growth. Toxoplasma increases host HK2 transcript and protein levels in a HIF-1-dependent manner. In addition, parasite growth at 3% oxygen is restored in HIF-1-deficient cells transfected with HK2 expression plasmids. Both HIF-1 activation and HK2 expression were accompanied by increases in host glycolytic flux, suggesting that enhanced HK2 expression in parasite-infected cells is functionally significant. Parasite dependence on host HK2 and HIF-1 expression is not restricted to transformed cell lines, as both are required for parasite growth in nontransformed C2C12 myoblasts and HK2 is upregulated in vivo following infection. While HK2 is normally associated with the cytoplasmic face of the outer mitochondrial membrane at physiological O2 levels, HK2 relocalizes to the host cytoplasm following infection, a process that is required for parasite growth at 3% oxygen. Taken together, our findings show that HIF-1-dependent expression and relocalization of HK2 represent a novel mechanism by which Toxoplasma establishes its replicative niche at tissue oxygen tensions.

Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

Three Wzy polymerases are specific for particular forms of an internal linkage in otherwise identical O units

The Wzx/Wzy-dependent pathway is the predominant pathway for O-antigen production in Gram-negative bacteria. The O-antigen repeat unit (O unit) is an oligosaccharide that is assembled at the cytoplasmic face of the membrane on undecaprenyl pyrophosphate. Wzx then flips it to the periplasmic face for polymerization by Wzy, which adds an O unit to the reducing end of a growing O-unit polymer in each round of polymerization. Wzx and Wzy both exhibit enormous sequence diversity. It has recently been shown that, contrary to earlier reports, the efficiency of diverse Wzx forms can be significantly reduced by minor structural variations to their native O-unit substrate. However, details of Wzy substrate specificity remain unexplored. The closely related galactose-initiated Salmonella O antigens present a rare opportunity to address these matters. The D1 and D2 O units differ only in an internal mannose-rhamnose linkage, and D3 expresses both in the same chain. D1 and D2 polymerases were shown to be specific for O units with their respective α or β configuration for the internal mannose-rhamnose linkage. The Wzy encoded by D3 gene cluster polymerizes only D1 O units, and deleting the gene does not eliminate polymeric O antigen, both observations indicating the presence of an additional wzy gene. The levels of Wzx and Wzy substrate specificity will affect the ease with which new O units can evolve, and also our ability to modify O antigens, capsules or secreted polysaccharides by glyco-engineering, to generate novel polysaccharides, as the Wzx/Wzy-dependent pathway is responsible for much of the diversity.