The GCP3-interacting proteins GIP1 and GIP2 are required for γ-tubulin complex protein localization, spindle integrity, and chromosomal stability

Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of γ-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with gamma-tubulin complex protein3 (GCP3), GCP3-interacting protein1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP-green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation.     

Minor contribution of presenilin 2 for γ-secretase activity in mouse embryonic fibroblasts and adult mouse brain

γ-Secretase plays an important function in the development of Alzheimer disease, since it participates in the production of the toxic amyloid β-peptide (Aβ) from the amyloid precursor protein (APP). Besides APP, γ-secretase cleaves many other substrates resulting in adverse side effects when γ-secretase inhibitors are used in clinical trials. γ-Secretase is a membrane bound protein complex consisting of at least four subunits, presenilin (PS), nicastrin, Aph-1 and Pen-2. PS and Aph-1 exist as different homologs (PS1/PS2 and Aph-1a/Aph-1b, respectively), which generates a variation in complex composition. PS1 and PS2 appears to have distinct roles since PS1 is essential during embryonic development whereas PS2 deficient mice are viable with a mild phenotype. The molecular mechanism behind this diversity is, however, largely unknown. In order to investigate whether PS1 and PS2 show different substrate specificity, we used PS1 or PS2 deficient mouse embryonic fibroblasts to study the processing on the γ-secretase substrates APP, Notch, N-cadherin, and ephrinB. We found that whereas depletion of PS1 severely affected the cleavage of all substrates, the effect of PS2 depletion was minor. In addition, less PS2 was found in active γ-secretase complexes. We also studied the effect of PS2 depletion in adult mouse brain and, in concordance with the results from the mouse embryonic fibroblasts, PS2 deficiency did not alter the cleavage of the two most important substrates, APP and Notch. In summary, this study shows that the contribution of PS2 on γ-secretase activity is of less importance, explaining the mild phenotype of PS2-deficient mice.     

Identification of amino acids involved in the binding of hMIP-1α to CC-CKR1, a MIP-lα receptor found on neutrophils

Human macrophage inflammatory protein-1 (hMIP-1) and human macrophage inflammatory protein-1 (hMIP-1) are chemokines involved in a diverse range of immunological effects. Both hMIP-1 and hMIP-1 are involved in the activation of monocytes and THP-1 cells probably through a common receptor(s). However, only hMIP-1 can bind to neutrophils with high affinity, presumably through CC-CKR1 (CKR1). Since the structure of these two proteins is highly conserved, non-conserved amino acids must define the disparate binding patterns that these two proteins exhibit. Measurements of binding, chemotaxis and calcium influx conducted with hMIP-1 and hMIP-1 chimeric proteins and mutants show that two amino acids (37K and 43L) are important in the binding and signaling of hMIP-1 through CKR1. Furthermore, we also show that mutations of the three charged amino acids at the C-terminus of hMIP-1 and hMIP-1 (amino acids 61, 65 and 67), do not adversely affect the binding to THP-1 cells.     

Changes in γ-Methyleneglutamic acid, γ-Methyleneglutamine and other amino acids and amides during fruit growth of Tribulus terrestris

Abstract

Distribution and metabolism of γ-methyleneglutamic acid, γ-methyleneglutamine and other amino acids and amides has been studied during fruit growth of Tribulus terrestris. The largest concentration of free amino acids and amides has been observed in fruit stage 1. The marked decline in the amount of γ-methyleneglutamic acid and γ-methyleneglutamine after fruit stage 1 may indicate their rapid utilization along with asparagine and glutamine during fruit growth. In leaf and in different fruit growth stages, γ-methyleneglutamic acid dominated over γ-methyleneglutamine.     

Sortilin, SorCS1b, and SorLA Vps10p sorting receptors, are novel γ-secretase substrates

Background

The mammalian Vps10p sorting receptor family is a group of 5 type I membrane homologs (Sortilin, SorLA, and SorCS1-3). These receptors bind various cargo proteins via their luminal Vps10p domains and have been shown to mediate a variety of intracellular sorting and trafficking functions. These proteins are highly expressed in the brain. SorLA has been shown to be down regulated in Alzheimer's disease brains, interact with ApoE, and modulate Aβ production. Sortilin has been shown to be part of proNGF mediated death signaling that results from a complex of Sortilin, p75^NTR and proNGF. We have investigated and provide evidence for γ-secretase cleavage of this family of proteins.

Results

We provide evidence that these receptors are substrates for presenilin dependent γ-secretase cleavage. γ-Secretase cleavage of these sorting receptors is inhibited by γ-secretase inhibitors and does not occur in PS1/PS2 knockout cells. Like most γ-secretase substrates, we find that ectodomain shedding precedes γ-secretase cleavage. The ectodomain cleavage is inhibited by a metalloprotease inhibitor and activated by PMA suggesting that it is mediated by an α-secretase like cleavage.

Conclusion

These data indicate that the α- and γ-secretase cleavages of the mammalian Vps10p sorting receptors occur in a fashion analogous to other known γ-secretase substrates, and could possibly regulate the biological functions of these proteins.     

Expression of a truncated form of the endoplasmic reticulum chaperone protein, σ1 receptor, promotes mitochondrial energy depletion and apoptosis

The σ1 receptor (σ(1)R) regulates endoplasmic reticulum (ER)/mitochondrial interorganellar Ca(2+) mobilization through the inositol 1,4,5-trisphosphate receptor (IP(3)R). Here, we observed that expression of a novel splice variant of σ(1)R, termed short form σ(1)R (σ(1)SR), has a detrimental effect on mitochondrial energy production and cell survival. σ(1)SR mRNA lacks 47 ribonucleotides encoding exon 2, resulting in a frameshift and formation of a truncated receptor. σ(1)SR localizes primarily in the ER at perinuclear regions and forms a complex with σ(1)R but not with IP(3)R in the mitochondrion-associated ER membrane. Overexpression of both σ(1)R and the truncated isoform promotes mitochondrial elongation with increased ER mitochondrial contact surface. σ(1)R overexpression increases the efficiency of mitochondrial Ca(2+) uptake in response to IP(3)R-driven stimuli, whereas σ(1)SR overexpression reduces it. Most importantly, σ(1)R promotes ATP production via increased mitochondrial Ca(2+) uptake, promoting cell survival in the presence of ER stress. By contrast, σ(1)SR suppresses ATP production following ER stress, enhancing cell death. Taken together, the newly identified σ(1)SR isoform interferes with σ(1)R function relevant to mitochondrial energy production under ER stress conditions, promoting cellular apoptosis.     

F-actin asymmetry and the endoplasmic reticulum–associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo

The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα–GPR–LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα–GPR–LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.     

HLA complex genes in type 1 diabetes and other autoimmune diseases. Which genes are involved?

The predisposition to develop a majority of autoimmune diseases is associated with specific genes within the human leukocyte antigen (HLA) complex. However, it is frequently difficult to determine which of the many genes of the HLA complex are directly involved in the disease process. The main reasons for these difficulties are the complexity of associations where several HLA complex genes might be involved, and the strong linkage disequilibrium that exists between the genes in this complex. The latter phenomenon leads to secondary disease associations, or what has been called 'hitchhiking polymorphisms'. Here, we give an overview of the complexity of HLA associations in autoimmune disease, focusing on type 1 diabetes and trying to answer the question: how many and which HLA genes are directly involved?     

Cdc37/Hsp90 protein-mediated regulation of IRE1α protein activity in endoplasmic reticulum stress response and insulin synthesis in INS-1 cells

IRE1α is an endoplasmic reticulum (ER) localized signaling molecule critical for unfolded protein response. During ER stress, IRE1α activation is induced by oligomerization and autophosphorylation in its cytosolic domain, a process triggered by dissociation of an ER luminal chaperone, binding immunoglobulin-protein (BiP), from IRE1α. In addition, inhibition of a cytosolic chaperone protein Hsp90 also induces IRE1α oligomerization and activation in the absence of an ER stressor. Here, we report that the Hsp90 cochaperone Cdc37 directly interacts with IRE1α through a highly conserved cytosolic motif of IRE1α. Cdc37 knockdown or disruption of Cdc37 interaction with IRE1α significantly increased basal IRE1α activity. In INS-1 cells, Hsp90 inhibition and disruption of IRE1α-Cdc37 interaction both induced an ER stress response and impaired insulin synthesis and secretion. These data suggest that Cdc37-mediated direct interaction between Hsp90/Cdc37 and an IRE1α cytosolic motif is important to maintain basal IRE1α activity and contributes to normal protein homeostasis and unfolded protein response under physiological stimulation.     

Determination of the proteolytic cleavage sites of the amyloid precursor-like protein 2 by the proteases ADAM10, BACE1 and γ-secretase

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development.     

Action of multiple endoplasmic reticulum chaperon-like proteins is required for proper folding and polarized localization of Kre6 protein essential in yeast cell wall β-1,6-glucan synthesis

Saccharomyces cerevisiae Kre6 is a type II membrane protein essential for cell wall β-1,6-glucan synthesis. Recently we reported that the majority of Kre6 is in the endoplasmic reticulum (ER), but a significant portion of Kre6 is found in the plasma membrane of buds, and this polarized appearance of Kre6 is required for β-1,6-glucan synthesis. An essential membrane protein, Keg1, and ER chaperon Rot1 bind to Kre6. In this study we found that in mutant keg1-1 cells, accumulation of Kre6 at the buds is diminished, binding of Kre6 to Keg1 is decreased, and Kre6 becomes susceptible to ER-associated degradation (ERAD), which suggests Keg1 participates in folding and transport of Kre6. All mutants of the calnexin cycle member homologues (cwh41, rot2, kre5, and cne1) showed defects in β-1,6-glucan synthesis, although the calnexin chaperon system is considered not functional in yeast. We found synthetic defects between them and keg1-1, and Cne1 co-immunoprecipitated with Keg1 and Kre6. A stronger binding of Cne1 to Kre6 was detected when two glucosidases (Cwh41 and Rot2) that remove glucose on N-glycan were functional. Skn1, a Kre6 homologue, was not detected by immunofluorescence in the wild type yeast, but in kre6Δ cells it became detectable and behaved like Kre6. In conclusion, the action of multiple ER chaperon-like proteins is required for proper folding and localization of Kre6 and probably Skn1 to function in β-1,6-glucan synthesis.     

Diverse pathways for maturation of the Na,K-ATPase β1 and β2 subunits in the endoplasmic reticulum of Madin-Darby canine kidney cells

Proper folding of the Na,K-ATPase β subunits followed by assembly with the α subunits is necessary for their export from the endoplasmic reticulum (ER). Here we examine roles of the ER lectin chaperone, calnexin, and non-lectin chaperone, BiP, in folding and quality control of the β(1) and β(2) subunits in Madin-Darby canine kidney cells. Short term prevention of glycan-calnexin interactions by castanospermine slightly increases ER retention of β(1), suggesting minor involvement of calnexin in subunit folding. However, both prolonged incubation with castanospermine and removal of N-glycosylation sites do not affect the α(1)-assembly or trafficking of β(1) but increase the amount of the β(1)-bound BiP, showing that BiP can compensate for calnexin in assisting β(1) folding. In contrast to β(1), prevention of either N-glycosylation or glycan-calnexin interactions abolishes the α(1)-assembly and export of β(2) from the ER despite increased β(2)-BiP binding. Mutations in the α(1)-interacting regions of β(1) and β(2) subunits impair α(1) assembly but do not affect folding of the β subunits tested by their sensitivity to trypsin. At the same time, these mutations increase the amount of β-bound BiP but not of β-bound calnexin and increase ER retention of both β-isoforms. BiP, therefore, prevents the ER export of folded but α(1)-unassembled β subunits. These α(1)-unassembled β subunits are degraded faster than α(1)-bound β subunits, preventing ER overload. In conclusion, folding of the β(1) and β(2) subunits is assisted predominantly by BiP and calnexin, respectively. Folded β(1) and β(2) either assemble with α(1) or bind BiP. The α(1)-bound β subunits traffic to the Golgi, whereas BiP-bound β subunits are retained and degraded in the ER.     

Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: discovery of PF-3084014

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aβ in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aβ EC₅₀ lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.     

The relation of protein binding to function: what is the significance of munc18 and synaptotagmin binding to syntaxin 1, and where are the corresponding binding sites?

The Q-SNARE syntaxin 1 is a central component of the synaptic membrane fusion machinery. Syntaxin probably interacts with multiple proteins during synaptic vesicle exocytosis. In vitro, the tightest binding partners for syntaxin 1 are other SNAREs (synaptobrevin/VAMP and SNAP-25) and munc18-1 (also known as rbsec1/nsec1). Recent studies on Drosophila syntaxin led to the surprising finding that a syntaxin mutant which does not bind the munc18-homolog Rop nevertheless functionally substitutes for wild-type syntaxin in membrane fusion (Wu et al., Neuron 23, 593-605, 1999). This observation suggested that syntaxin 1 binding to munc18-1 is not essential for fusion, a puzzling conclusion in view of the tight binding of munc18 and syntaxin homologs in all organisms. To address this issue, we have now reinvestigated the interaction of syntaxin with munc18 and Rop. We find that the syntaxin sequence that was mutated in the Drosophila studies is not essential for munc18/Rop binding, and that the mutant is in fact able to bind to munc18/Rop. Thus the fact that the mutant syntaxin rescues release cannot be used as an argument that munc18 binding is not essential. In addition to munc18 and SNAREs, several other proteins have been suggested to interact with various domains of syntaxin 1, notably the calcium-sensor synaptotagmin and the vesicle protein CSP. Our results confirm that the SNARE motif in syntaxin binds to synaptotagmin, but this interaction does not require the very C-terminus of the motif. Interestingly, binding of synaptotagmin appears to be decreased in the closed conformation of syntaxin. In contrast, no interaction of CSP with syntaxin was detected even under low-stringency conditions. Our data suggest 1., that assays measuring protein/protein interactions that involve syntaxin may be more difficult to evaluate than is often assumed because of the sticky nature of the proteins involved, and 2., that it is currently not possible to draw conclusions about the importance of the various interactions with the available data from Drosophila or vertebrates.     

Internalisation and multiple phosphorylation of γ-Conglutin,the lupin seed glycaemia-lowering protein,in HepG2 cells

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action.     

Effects of δ-aminolaevulinic acid,porphobilinogen and structurally related amino acids on 2-deoxyglucose uptake in cultured neurons

The effects of -aminolaevulinic acid (ALA), porphobilinogen (PBG), -aminobutyric acid (GABA), muscimol, glutamic acid and kainic acid on [³H]2-deoxy-d-glucose uptake by cultured neurons were investigated. Exposure of the cultures for 4 days, to ALA at concentrations as low as 10 M caused a significant, dose-dependent decrease in [³H]2-deoxy-d-glucose uptake. Neither ALA nor PBG appeared to interfere directly with glucose transport into the neuron but 1 mM ALA caused an initial stimulation of [³H]2-deoxy-d-glucose uptake which increased to a maximum after 4 hr and fell to below control values after 19 hr exposure. GABA and muscimol caused similar increases in [³H]2-deoxy-d-glucose uptake but these values remained above control levels after 19 hr exposure. Glutamic acid and kainic acid caused an immediate increase in [³H]2-deoxy-d-glucose uptake which declined to mininum values after 4 hr exposure. The effect of ALA on glucose utilization in neurons may be of particular relevance to patients with acute porphyria where a genetic lesion in neural haem and haemoprotein biosynthesis is postulated to occur. ALA appeared to be more toxic to the neurons than any of the other compounds tested, possibly causing a critical depletion of energy reserves and cell death.     

Glycosylation, transport, and complex formation of palmitoyl protein thioesterase 1 (PPT1) – distinct characteristics in neurons

Background

About 20 % of nemaline myopathies are thus far related to skeletal muscle alpha-actin. Seven actin mutants located in different parts of the actin molecule and linked to different forms of the disease were selected and expressed as EGFP-tagged constructs in differentiated C2C12 mytoubes. Results were compared with phenotypes in patient skeletal muscle fibres and with previous expression studies in fibroblasts and C2C12 myoblasts/myotubes.

Results

Whereas EGFP wt-actin nicely incorporated into endogenous stress fibres and sarcomeric structures, the mutants showed a range of phenotypes, which generally changed upon differentiation. Many mutants appeared delocalized in myoblasts but integrated into endogenous actin structures after 4–6 days of differentiation, demonstrating a poor correlation between the appearance in myotubes and the severity of the disease. However, for some mutants, integration into stress fibres induced aberrant structures in differentiated cells, like thickening or fragmentation of stress fibres. Other mutants almost failed to integrate but formed huge aggregates in the cytoplasm of myotubes. Those did not co-stain with alpha-actinin, a main component of nemaline bodies found in patient muscle. Interestingly, nuclear aggregates as formed by two of the mutants in myoblasts were found less frequently or not at all in differentiated cells.

Conclusion

Myotubes are a suitable system to study the capacity of a mutant to incorporate into actin structures or to form or induce pathological changes. Some of the phenotypes observed in undifferentiated myoblasts may only be in vitro effects. Other phenotypes, like aberrant stress fibres or rod formation may be more directly correlated with disease phenotypes. Some mutants did not induce any changes in the cellular actin system, indicating the importance of additional studies like functional assays to fully characterize the pathological impact of a mutant.     

The effect of charge reversal mutations in the α-helical region of liver fatty acid binding protein on the binding of fatty-acyl CoAs,lysophospholipids and bile acids

Liver fatty acid binding protein (LFABP) is unique among the various types of FABPs in that it can bind a variety of ligands in addition to fatty acids. LFABP is able to bind long chain fatty acids with a 2:1 stoichiometry and the crystal structure has identified two fatty acid binding sites in the binding cavity. The presumed primary site (site 1) involves the fatty acid binding with the carboxylate group buried in the cavity whereas the fatty acid at site 2 has the carboxylate group solvent-exposed within the ligand portal region and in the vicinity of -helix II. The -helical region contains three cationic residues, K20, K31, K33 and modelling studies suggest that K31 on -helix II could make an electrostatic contribution to anionic ligands binding to site 2. The preparation of three charge reversal mutants of LFABP, K20E, K31E and K33E has allowed an investigation of the role of site 2 in ligand binding, particularly those ligands with a bulky anionic head group. The binding of oleoyl CoA, lysophosphatidic acid, lysophosphatidylcholine, lithocholic acid and taurolithocholate 3-sulphate to LFABP has been studied using the -helical mutants. The results support the concept that such ligands bind at site 2 of LFABP where solvent exposure allows the accommodation of their bulky anionic group.