We have found that the mammalian Ran GTPase-activating protein RanGAP1 is highly concentrated at the cytoplasmic periphery of the nuclear pore complex (NPC), where it associates with the 358-kDa Ran-GTP-binding protein RanBP2. This interaction requires the ATP-dependent posttranslational conjugation of RanGAP1 with SUMO-1 (for small ubiquitin-related modifier), a novel protein of 101 amino acids that contains low but significant homology to ubiquitin. SUMO-1 appears to represent the prototype for a novel family of ubiquitin-related protein modifiers. Inhibition of nuclear protein import resulting from antibodies directed at NPC-associated RanGAP1 cannot be overcome by soluble cytosolic RanGAP1, indicating that GTP hydrolysis by Ran at RanBP2 is required for nuclear protein import.
SUMMARY G protein-coupled receptors form hetero-dimers and higher order hetero-oligomers, yet the significance of receptor heteromerization in cellular and behavioral responses is poorly understood. Atypical antipsychotic drugs, such as clozapine and risperidone all have in common a high affinity for the serotonin 5-HT2A receptor (2AR). However, closely related nonantipsychotic drugs, such as ritanserin and methysergide, while blocking 2AR function, lack comparable neuropsychological effects. Why some but not all drugs that inhibit 2AR-dependent signaling exhibit antipsychotic properties remains unresolved. We found that a heteromeric complex formed between the metabotropic glutamate 2 receptor (mGluR2) and the 2AR critically integrates the action of drugs affecting signaling and behavioral outcomes. Acting through the mGluR2/2AR heterocomplex, both glutamatergic and serotonergic drugs achieve a balance between Gi- and Gq-dependent signaling that predicts their psychoactive behavioral effects. These observations provide a novel mechanistic insight into antipsychotic action that may advance therapeutic strategies for schizophrenia.
The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.
Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1, 2). In Xenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with Ran-GAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.The transport of selected proteins and nucleic acids across the nuclear pore complex (NPC) 1 is regulated by the small GTPase Ran/TC4 (4, 5). Ran alternates between a GTP-bound state and a GDP-bound state, a transition facilitated by a nuclear GTP-exchange factor (RCC1) and a cytoplasmic GTPase-activating protein (RanGAP1) (reviewed in Refs. 6 and 7). The differential binding of Ran-GTP and Ran-GDP to carrier proteins (e.g. importins, transportin, and NTF2) and NPC proteins provides a mechanism that allows Ran to regulate the transport of cargo across the nuclear pore complex. In this system, the subcellular localization of RCC1 and RanGAP1 are major determinants of directed nuclear transport.The localization of RanGAP1 to the nuclear pore complex is imparted by its specific association with Nup358/RanBP2 (1), a component of the cytoplasmic filaments emanating from the NPC (8, 9). This interaction requires the covalent modification of RanGAP1 with a ubiquitin-related protein designated SUMO-1 (1, 2). Recently, Ubc9, a structural homologue of the E2-type UBCs, was shown to coimmunoprecipitate with Nup358 in Xenopus egg extracts (3). Previous studies indicated that Ubc9 may be required for regulating the progression through the cell cycle in yeast (10,11). In addition, the human homologue of Ubc9 (HsUbc9) can functionally substitute for yeast Ubc9 (12,13). Although HsUbc9 has been proposed to mediate the ubiquitin-catalyzed degradation of mitotic cyclins (10 -12), no bona fide ubiquitin-conjugating activity has been demonstrated for HsUbc9.We now show that HsUbc9 differs significantly from the E2 family of UBCs in that it can form a thiolester intermediate with SUMO-1, rather than ubiquitin. This enzymatic activity is ATP-dependent and is not observed in mutants of HsUbc9, which contain a single amino acid substitution at the putative catalytic site. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. MATERIALS AND METHODS Preparation of Affinity Purified Antibodies-Rabbit antiserum raisedagainst Escherichia coli-derived recombinant HsUbc9 was affinity purified by passage over a CNBr-activated Sepharose B matrix coupled to E. coli-deri...
Objective. To describe the clinical profile, microbiological aetiologies, and management outcomes in patients with liver abscess. Methods. A cross-sectional study was conducted from May, 2011, to April, 2013, on 200 consecutive liver abscess patients at PGIMER and Dr. RML Hospital, New Delhi. History, examination, and laboratory investigations were recorded. Ultrasound guided aspiration was done and samples were investigated. Chi-square test and multivariate regression analysis were performed to test association. Results. The mean age of patients was 41.13 years. Majority of them were from lower socioeconomic class (67.5%) and alcoholic (72%). The abscesses were predominantly in right lobe (71%) and solitary (65%). Etiology of abscess was 69% amoebic, 18% pyogenic, 7.5% tubercular, 4% mixed, and 1.5% fungal. Percutaneous needle aspiration was done in 79%, pigtail drainage in 17%, and surgical intervention for rupture in 4% patients. Mortality was 2.5%, all reported in surgical group. Solitary abscesses were amoebic and tubercular whereas multiple abscesses were pyogenic (P = 0.001). Right lobe was predominantly involved in amoebic and pyogenic abscesses while in tubercular abscesses left lobe involvement was predominant (P = 0.001). Conclusions. The commonest presentation was young male, alcoholic of low socioeconomic class having right lobe solitary amoebic liver abscess. Appropriate use of minimally invasive drainage techniques reduces mortality.
The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP2 can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP2-dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP2 interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP2 levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP2 promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease.
Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca2+]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states.
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