The Interactome of the VAP Family of Proteins: An Overview

Membrane contact sites (MCS) are sites of close apposition of two organelles that help in lipid transport and synthesis, calcium homeostasis and several other biological processes. The VAMP-associated proteins (VAPs) VAPA, VAPB, MOSPD2 and the recently described MOSPD1 and MOSPD3 are tether proteins of MCSs that are mainly found at the endoplasmic reticulum (ER). VAPs interact with various proteins with a motif called FFAT (two phenylalanines in an acidic tract), recruiting the associated organelle to the ER. In addition to the conventional FFAT motif, the recently described FFNT (two phenylalanines in a neutral tract) and phospho-FFAT motifs contribute to the interaction with VAPs. In this review, we summarize and compare the recent interactome studies described for VAPs, including in silico and proximity labeling methods. Collectively, the interaction repertoire of VAPs is very diverse and highlights the complexity of interactions mediated by the different FFAT motifs to the VAPs.

Functional Organization of the Endoplasmic Reticulum Dictates the Susceptibility of Target Cells to Arsenite-Induced Mitochondrial Superoxide Formation, Mitochondrial Dysfunction and Apoptosis

Arsenite induces many critical effects associated with the formation of reactive oxygen species (ROS) through different mechanisms. We focused on the Ca2+-dependent mitochondrial superoxide (mitoO2-.) formation and addressed questions on the effects of low concentrations of arsenite on the mobilization of the cation from the endoplasmic reticulum and the resulting mitochondrial accumulation. Using various differentiated and undifferentiated cell types uniquely expressing the inositol-1, 4, 5-triphosphate receptor (IP3R), or both the IP3R and the ryanodine receptor (RyR), we determined that expression of this second Ca2+ channel is an absolute requirement for mitoO2-. formation and for the ensuing mitochondrial dysfunction and downstream apoptosis. In arsenite-treated cells, RyR was recruited after IP3R stimulation and agonist studies indicated that in these cells RyR is in close apposition with mitochondria. It was also interesting to observe that arsenite fails to promote mitochondrial Ca2+ accumulation, mitoO2-. formation, mitochondrial toxicity in RyR-devoid cells, in which the IP3R is in close contact with the mitochondria. We therefore conclude that low dose arsenite-induced mitoO2- formation and the resulting mitochondrial dysfunction and toxicity, are prerequisite of cell types expressing the RyR in close apposition with mitochondria.

The Fast and the Furious: Golgi Contact Sites

Contact sites are areas of close apposition between two membranes that coordinate nonvesicular communication between organelles. Such interactions serve a wide range of cellular functions from regulating metabolic pathways to executing stress responses and coordinating organelle inheritance. The past decade has seen a dramatic increase in information on certain contact sites, mostly those involving the endoplasmic reticulum. However, despite its central role in the secretory pathway, the Golgi apparatus and its contact sites remain largely unexplored. In this review, we discuss the current knowledge of Golgi contact sites and share our thoughts as to why Golgi contact sites are understudied. We also highlight what exciting future directions may exist in this emerging field.

Novel insights into the metabolic action of Kiss1 neurons

Kiss1 neurons are essential regulators of the hypothalamic–pituitary–gonadal (HPG) axis by regulating gonadotropin-releasing hormone (GnRH) release. Compelling evidence suggests that Kiss1 neurons of the arcuate nucleus (Kiss1ARC), recently identified as the hypothalamic GnRH pulse generator driving fertility, also participate in the regulation of metabolism through kisspeptinergic and glutamatergic interactions with, at least, proopiomelanocortin (POMC) and agouti-related peptide (AgRP)/neuropeptide Y (NPY) neurons, located in close apposition with Kiss1ARC. This review offers a comprehensive overview of the recent developments, mainly derived from animal models, on the role of Kiss1 neurons in the regulation of energy balance, including food intake, energy expenditure and the influence of circadian rhythms on this role. Furthermore, the possible neuroendocrine pathways underlying this effect, and the existing controversies related to the anorexigenic action of kisspeptin in the different experimental models, are also discussed.

Contacting domains that segregate lipid from solute transporters in malaria parasites

ABSTRACTWhile membrane contact sites (MCS) between intracellular organelles are abundant1, and cell-cell junctions are classically defined2, very little is known about the contacts between membranes that delimit extracellular junctions within cells, such as those of chloroplasts and intracellular parasites. The malaria parasite replicates within a unique organelle, the parasitophorous vacuole (PV) but the mechanism(s) are obscure by which the limiting membrane of the PV, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the host erythrocyte (RBC). Here, we demonstrate the existence of multiple micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localized the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We found that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. Thus, these two different transport systems handling hydrophilic and hydrophobic substances, respectively, assume complementary and exclusive distributions. This new structural and molecular data assigns a functional significance to a macroscopic membrane domain.

Neurodevelopmental mutation of giant ankyrin-G disrupts a core mechanism for axon initial segment assembly

Giant ankyrin-G (gAnkG) coordinates assembly of axon initial segments (AISs), which are sites of action potential generation located in proximal axons of most vertebrate neurons. Here, we identify a mechanism required for normal neural development in humans that ensures ordered recruitment of gAnkG and β4-spectrin to the AIS. We identified 3 human neurodevelopmental missense mutations located in the neurospecific domain of gAnkG that prevent recruitment of β4-spectrin, resulting in a lower density and more elongated pattern for gAnkG and its partners than in the mature AIS. We found that these mutations inhibit transition of gAnkG from a closed configuration with close apposition of N- and C-terminal domains to an extended state that is required for binding and recruitment of β4-spectrin, and normally occurs early in development of the AIS. We further found that the neurospecific domain is highly phosphorylated in mouse brain, and that phosphorylation at 2 sites (S1982 and S2619) is required for the conformational change and for recruitment of β4-spectrin. Together, these findings resolve a discrete intermediate stage in formation of the AIS that is regulated through phosphorylation of the neurospecific domain of gAnkG.

JAK2 V617F Dimerizes Homodimeric Cytokine Receptors Cytosolic Domains By Requiring Pseudokinase Domain Residues That Promote JAK2 Dimerization and Oncogenic Activity

JAK2 plays roles in several signaling pathways by binding to multiple distinct cytokine receptors. Dysfunction of JAK2 signaling is associated with hematopoietic malignancies. The acquired mutation JAK2 V617F, which constitutively activates JAK2 kinase, is the most common molecular event associated with myeloproliferative disorders. Using a combination of bioluminescence energy transfer and protein fragment complementation assays we show that V617F located in the pseudokinase domain of JAK2 plays an important role on JAK2 dimerization via the C-terminal kinase domains. Using NanoBRET we show that JAK2 dimerization via the C-terminal domains is reduced by single mutations (E596R 1, F595A 2, A598F and F537A) that in the context of JAK2 V617F revert the mutant to wild type phenotype with respect to signaling by homodimeric erythropoietin and thrombopoietin receptors (EpoR and TpoR). Thus, residues that are required for V617F activation strengthen JAK2 dimerization via C-terminal kinase domains, implicating the pseudokinase domain in global JAK2 dimerization and suggesting that part of the V617F activation mechanism relies on dimerization of mutated pseudokinase domains. Using NanoBiT protein fragment complementation assay, we explored the effects of JAK2 V617F on dimerization of the cytosolic ends of cytokine receptors. We show that JAK2 V617F promotes strong enhancement of dimerization of EpoR and TpoR cytosolic domains, when compared to wild type JAK2. This result is unexpected for EpoR that was shown to exist in a preformed dimer configuration at a significantly higher degree than TpoR3-4. Nevertheless our results indicate that JAK2 V617F enhances close apposition of cytosolic domains of EpoR and TpoR, which could occur within preformed complexes or by a switch from monomer to dimer configuration induced by JAK2 V671F. This enhancement of dimerization is prevented when JAK2 V617F carries any of the reverting mutations (E596R, F595A, A598F, F537A) that prevent V617F activation. We also detected enhanced EpoR dimerization for exon 12 JAK2 mutation and JAK2 T875N, but not for other JAK2 mutants that are involved in lymphoid malignancies. The signals we detect are not simply the result of signaling and clustering in vesicles or internalization, as inhibition of signaling via Ruxolitinib does not prevent the dimerization effects on receptors. Our results indicate that rather than preformed dimeric receptors aiding in JAK2 V617F activation5, JAK2 V617F promotes close apposition of cytosolic domains of dimeric receptors. This result is in agreement with a recent study that showed that JAK2 V617F requires a closer proximity of EpoR chains than signaling by wild type JAK26. Such differences in dimeric requirements might open new therapeutic avenues for JAK2 V617F inhibition. We extended these explorations to other cytokine receptors utilizing JAK2 or JAK1 belonging to type I and type II subfamilies. Results on JAK2 are transposable to JAK1 for homologous mutations. Most receptors tested, such as EpoR, TpoR, G-CSFR, IL2, IL9 and type I interferon respond to their cytokine when JAK2 (or JAK1 for those utilizing JAK1) carries mutations known to inhibit JAK2 V617F (or JAK1 V658F, homologous to JAK2 V617F). One prominent exception is interferon γ-receptor (IFNGR), which is significantly impaired in its response to IFNγ when JAK2 and JAK1 carry such mutations. We show that homodimeric interactions within the heterotetrameric IFNGR recruiting JAK2 and JAK1 explain this inhibitory effect. Thus, it appears that a specific region around pseudokinase domain helix αC, which is required for JAK2 V617F and JAK1 V658F oncogenic activation, is also critical for relaying normal IFNγ signaling via its tetrameric receptor complex, which appears to require enhanced dimerization between JAK1 and JAK2 pseudokinase domains. Our results provide critical information for the design of strategies aiming to inhibit JAK2 V617F and JAK1 V658F, and also provide new avenues to select for compounds able to prevent excessive IFNγ signaling, which is present in certain autoimmune conditions.Leroy, E.; et al. Biochem J2016,473 (11), 1579-91.Dusa, A.; et al. PLoS One2010,5 (6), e11157.Defour, J. P.; et al. Proc Natl Acad Sci U S A2013,110 (7), 2540-5.Constantinescu, S. N.; et al. Proc Natl Acad Sci U S A2001,98 (8), 4379-84.Lu, X.; et al. Proc Natl Acad Sci U S A2005,102 (52), 18962-7.Moraga, I.; et al. Cell2015,160 (6), 1196-208. Disclosures Constantinescu: Novartis: Membership on an entity's Board of Directors or advisory committees; Personal Genetics: Membership on an entity's Board of Directors or advisory committees; MyeloPro Research and Diagnostics GmbH: Equity Ownership; Novartis: Consultancy; AlsaTECH: Equity Ownership; Novartis: Honoraria.

Manner-of-speaking that-complements as close apposition structures

An elusive property of that-clauses following manner-of-speaking verbs is that they do not behave like that-clauses following other non-factive verbs when it comes to the availability of wh-extraction, main clause phenomena and complementizer drop. Non-factive that-clauses allow wh-extraction, main clause phenomena and complementizer drop, but manner-of-speaking that-clauses resist them. In addition, the behavior of manner-of-speaking that-clauses patterns with noun complement clauses and that-clauses following the pronoun it. In this paper, I argue that the referential and adjunct status of manner-of-speaking that-clauses, noun complement clauses and that-clauses following the pronoun it is responsible for their shared restrictions on wh-extraction, main clause phenomena and complementizer drop. Specifically, I argue all three of these that-clauses are referential adjuncts in a close apposition relationship with a nominal object.