Insoluble Cellular Fraction Research Articles

Novel loss of function mutation in TUBA1A gene compromises tubulin stability and proteostasis causing spastic paraplegia and ataxia.

Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for α and β-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as "tubulinopathies" and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance. Although tubulin mutations have been classically linked to neurodevelopmental defects, growing evidence demonstrates that perturbations of tubulin functions and activities may also drive neurodegeneration. In this study, we causally link the previously unreported missense mutation p.I384N in TUBA1A, one of the neuron-specific α-tubulin isotype I, to a neurodegenerative disorder characterized by progressive spastic paraplegia and ataxia. We demonstrate that, in contrast to the p.R402H substitution, which is one of the most recurrent TUBA1A pathogenic variants associated to lissencephaly, the present mutation impairs TUBA1A stability, reducing the abundance of TUBA1A available in the cell and preventing its incorporation into microtubules. We also show that the isoleucine at position 384 is an amino acid residue, which is critical for α-tubulin stability, since the introduction of the p.I384N substitution in three different tubulin paralogs reduces their protein level and assembly into microtubules, increasing their propensity to aggregation. Moreover, we demonstrate that the inhibition of the proteasome degradative systems increases the protein levels of TUBA1A mutant, promoting the formation of tubulin aggregates that, as their size increases, coalesce into inclusions that precipitate within the insoluble cellular fraction. Overall, our data describe a novel pathogenic effect of p.I384N mutation that differs from the previously described substitutions in TUBA1A, and expand both phenotypic and mutational spectrum related to this gene.

Folylpoly-ɣ-glutamate synthetase association to the cytoskeleton: Implications to folate metabolon compartmentalization

Folates are essential for nucleotide biosynthesis, amino acid metabolism and cellular proliferation. Following carrier-mediated uptake, folates are polyglutamylated by folylpoly-ɣ-glutamate synthetase (FPGS), resulting in their intracellular retention. FPGS appears as a long isoform, directed to mitochondria via a leader sequence, and a short isoform reported as a soluble cytosolic protein (cFPGS). However, since folates are labile and folate metabolism is compartmentalized, we herein hypothesized that cFPGS is associated with the cytoskeleton, to couple folate uptake and polyglutamylation and channel folate polyglutamates to metabolon compartments. We show that cFPGS is a cytoskeleton-microtubule associated protein: Western blot analysis revealed that endogenous cFPGS is associated with the insoluble cellular fraction, i.e., cytoskeleton and membranes, but not with the cytosol. Mass spectrometry analysis identified the putative cFPGS interactome primarily consisting of microtubule subunits and cytoskeletal motor proteins. Consistently, immunofluorescence microscopy with cytosol-depleted cells demonstrated the association of cFPGS with the cytoskeleton and unconventional myosin-1c. Furthermore, since anti-microtubule, anti-actin cytoskeleton, and coatomer dissociation-inducing agents yielded perinuclear pausing of cFPGS, we propose an actin- and microtubule-dependent transport of cFPGS between the ER-Golgi and the plasma membrane. These novel findings support the coupling of folate transport with polyglutamylation and folate channeling to intracellular metabolon compartments. SignificanceFPGS, an essential enzyme catalyzing intracellular folate polyglutamylation and efficient retention, was described as a soluble cytosolic enzyme in the past 40 years. However, based on the lability of folates and the compartmentalization of folate metabolism and nucleotide biosynthesis, we herein hypothesized that cytoplasmic FPGS is associated with the cytoskeleton, to couple folate transport and polyglutamylation as well as channel folate polyglutamates to biosynthetic metabolon compartments. Indeed, using complementary techniques including Mass-spectrometry proteomics and fluorescence microscopy, we show that cytoplasmic FPGS is associated with the cytoskeleton and unconventional myosin-1c. This novel cytoskeletal localization of cytoplasmic FPGS supports the dynamic channeling of polyglutamylated folates to metabolon compartments to avoid oxidation and intracellular dilution of folates, while enhancing folate-dependent de novo biosynthesis of nucleotides and DNA/protein methylation.

Alpha-synuclein suppresses mitochondrial protease ClpP to trigger mitochondrial oxidative damage and neurotoxicity

Both α-Synuclein (αSyn) accumulation and mitochondrial dysfunction have been implicated in the pathology of Parkinson’s disease (PD). Although studies suggest that αSyn and its missense mutant, A53T, preferentially accumulate in the mitochondria, the mechanisms by which αSyn and mitochondrial proteins regulate each other to trigger mitochondrial and neuronal toxicity are poorly understood. ATP-dependent Clp protease (ClpP), a mitochondrial matrix protease, plays an important role in regulating mitochondrial protein turnover and bioenergetics activity. Here, we show that the protein level of ClpP is selectively decreased in αSyn-expressing cell culture and neurons derived from iPS cells of PD patient carrying αSyn A53T mutant, and in dopaminergic (DA) neurons of αSyn A53T mice and PD patient postmortem brains. Deficiency in ClpP induces an overload of mitochondrial misfolded/unfolded proteins, suppresses mitochondrial respiratory activity, increases mitochondrial oxidative damage and causes cell death. Overexpression of ClpP reduces αSyn-induced mitochondrial oxidative stress through enhancing the level of Superoxide Dismutase-2 (SOD2), and suppresses the accumulation of αSyn S129 phosphorylation and promotes neuronal morphology in neurons derived from PD patient iPS cells carrying αSyn A53T mutant. Moreover, we find that αSyn WT and A53T mutant interact with ClpP and suppress its peptidase activity. The binding of αSyn to ClpP further promotes a distribution of ClpP from soluble to insoluble cellular fraction in vitro and in vivo, leading to reduced solubility of ClpP. Compensating for the loss of ClpP in the substantia nigra of αSyn A53T mice by viral expression of ClpP suppresses mitochondrial oxidative damage, and reduces αSyn pathology and behavioral deficits of mice. Our findings provide novel insights into the mechanism underlying αSyn-induced neuronal pathology, and they suggest that ClpP might be a useful therapeutic target for PD and other synucleinopathies.

Dimer‐specific immunoprecipitation of active caspase‐2 identifies TRAF proteins as novel activators

Caspase‐2 has been shown to initiate apoptotic cell death in response to specific intracellular stressors such as DNA damage. However, the molecular mechanisms immediately upstream of its activation are still poorly understood. We combined a caspase‐2 bimolecular fluorescence complementation (BiFC) system with fluorophore‐specific immunoprecipitation to isolate and study the active caspase‐2 dimer and its interactome. Using this technique, we found that tumor necrosis factor receptor‐associated factor 2 (TRAF2), as well as TRAF1 and 3, directly binds to the active caspase‐2 dimer. TRAF2 in particular is necessary for caspase‐2 activation in response to apoptotic cell death stimuli. Furthermore, we found that dimerized caspase‐2 is ubiquitylated in a TRAF2‐dependent manner at K15, K152, and K153, which in turn stabilizes the active caspase‐2 dimer complex, promotes its association with an insoluble cellular fraction, and enhances its activity to fully commit the cell to apoptosis. Together, these data indicate that TRAF2 positively regulates caspase‐2 activation and consequent cell death by driving its activation through dimer‐stabilizing ubiquitylation.

ULK1-mediated phosphorylation of ATG14 promotes autophagy and is impaired in Huntington's disease models.

BackgroundAutophagy is a bulk degradation pathway for long-lived proteins, protein aggregates, and damaged organelles. ULK1 protein kinase and Vps34 lipid kinase are two key autophagy regulators that are critical for autophagosome biogenesis. However, it isn’t fully understood how ULK1 regulates Vps34, especially in the context of disease. Polyglutamine expansion in huntingtin (Htt) causes aberrant accumulation of the aggregated protein and disrupts various cellular pathways including autophagy, a lysosomal degradation pathway, underlying the pathogenesis of Huntington’s disease (HD). Although autophagic clearance of Htt aggregates is under investigation as therapeutic strategy for HD, the precise mechanism of autophagy impairment remains poorly understood. Moreover, in-vivo assays of autophagy have been particularly challenging due to lack of reliable and robust molecular biomarkers.MethodWe generated anti-phosphorylated ATG14 antibody to determine ATG14-mediated autophagy regulation; we employed Huntington’s disease (HD) genetic cell models and animal models as well as autophagy reporter animal model to understand autophagy signaling and regulation in vivo. We applied biochemical analysis and molecular biology approaches to dissect the alteration of autophagy kinase activity and regulation.ResultsHere, we demonstrate that ULK1 phosphorylates ATG14 at serine 29 in an mTOR-dependent manner. This phosphorylation critically regulates ATG14-Vps34 lipid kinase activity to control autophagy level. We also show that ATG14-associated Vps34 activity and ULK1-mediated phosphorylation of ATG14 and Beclin 1 are compromised in the Q175 mouse model of Huntington’s disease. Finally, we show that ATG14 phosphorylation is decreased during general proteotoxic stress caused by proteasomal inhibition. This reduction of the specific phosphorylation of ATG14 and Beclin 1 is mediated, in part, by p62-induced sequestration of ULK1 to an insoluble cellular fraction. We show that increased ULK1 levels and phosphor-mimetic mutant ATG14 facilitate the clearance of polyQ mutant in cells.ConclusionOur study identifies a new regulatory mechanism for ATG14-Vps34 kinase activity by ULK1, which can be used as valuable molecular markers for in-vivo autophagic activity as well as potential therapeutic target for the clearance of polyglutamine disease protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0141-0) contains supplementary material, which is available to authorized users.

IL-10 Alters Immunoproteostasis in APP Mice, Increasing Plaque Burden and Worsening Cognitive Behavior

Anti-inflammatory strategies are proposed to have beneficial effects in Alzheimer's disease. To explore how anti-inflammatory cytokine signaling affects Aβ pathology, we investigated the effects of adeno-associated virus (AAV2/1)-mediated expression of Interleukin (IL)-10 in the brains of APP transgenic mouse models. IL-10 expression resulted in increased Aβ accumulation and impaired memory in APP mice. A focused transcriptome analysis revealed changes consistent with enhanced IL-10 signaling and increased ApoE expression in IL-10-expressing APP mice. ApoE protein was selectively increased in the plaque-associated insoluble cellular fraction, likely because of direct interaction with aggregated Aβ in the IL-10-expressing APP mice. Ex vivo studies also show that IL-10 and ApoE can individually impair glial Aβ phagocytosis. Our observations that IL-10 has an unexpected negative effect on Aβ proteostasis and cognition in APP mouse models demonstrate the complex interplay between innate immunity and proteostasis in neurodegenerative diseases, an interaction we call immunoproteostasis.

Changing partners: transcription factors form different complexes on and off chromatin.

The current knowledge on how protein–protein interactions regulate the function of transcription factors (TFs) has remained limited due to an incomplete knowledge of their interaction partners. In their recent work, Chen and colleagues (Li et al, 2015) analyse the interactome of 56 TFs and reveal distinct chromatin-associated and soluble TF complexes.

CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a “difficult-to-express” immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate

Silver (Ag) possesses antibacterial activity and has been used in wound dressings and deodorant powders worldwide. However, the metabolic behavior and biological roles of Ag in mammals have not been well characterized. In the present study, we exposed human bronchial epithelial cells (BEAS-2B) to AgNO3 and investigated uptake and intracellular distribution of Ag, expression of metallothionein (MT), generation of reactive oxygen species (ROS), and changes in mitochondrial respiration. The culture medium concentration of Ag decreased with time and stabilized at 12h. The concentration of both Ag and MT in the soluble cellular fraction increased up to 3h and then decreased, indicating that cytosolic Ag relocated to the insoluble fraction of the cells. The levels of mRNAs for the major human MT isoforms MT-I and MT-II paralleled with the protein levels of Ag-MT. The intensity of fluorescence derived from ROS was elevated in the mitochondrial region at 24h. Ag decreased mitochondrial oxygen consumption in a dose-dependent manner and the activity of mitochondrial complex I-IV enzymes was significantly inhibited following exposure to Ag. In a separate experiment, we found that hydrogen peroxide (H2O2) at concentrations as low as 0.001% (equivalent to the concentration of H2O2 in Ag-exposed cells) removed Ag from MT. These results suggest MT was decomposed by cytosolic H2O2, and then Ag released from MT relocated to insoluble cellular fractions and inhibited electron chain transfer of mitochondrial complexes, which eventually led to cell damage.

Effective enhancement of Pseudomonas stutzeri D-phenylglycine aminotransferase functional expression in Pichia pastoris by co-expressing Escherichia coli GroEL-GroES

BackgroundD-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT.ResultsIntracellular expression of the codon-optimized synthetic dpgA gene under the PAOX1 promoter in P. pastoris resulted in inactive D-PhgAT associated with insoluble cellular fraction and very low level of D-PhgAT activity in the soluble fraction. Manipulation of culture conditions such as addition of sorbitol to induce intracellular accumulation of osmolytes, addition of benzyl alcohol to induce chaperone expression, or lowering incubation temperature to slow down protein expression and folding rates all failed to increase the active D-PhgAT yield. Co-expression of E. coli chaperonins GroEL-GroES with the D-PhgAT dramatically improved the soluble active enzyme production. Increasing gene dosage of both the dpgA and those of the chaperones further increased functional D-PhgAT yield up to 14400-fold higher than when the dpgA was expressed alone. Optimization of cultivation condition further increased D-PhgAT activity yield from the best co-expressing strain by 1.2-fold.ConclusionsThis is the first report on the use of bacterial chaperones co-expressions to enhance functional intracellular expression of bacterial enzyme in P. pastoris. Only two bacterial chaperone genes groEL and groES were sufficient for dramatic enhancement of functionally active D-PhgAT expression in this yeast. With the optimized gene dosage and chaperone combinations, P. pastoris can be attractive for intracellular expression of bacterial proteins since it can grow to a very high cell density which is translated into the higher volumetric product yield than the E. coli or other bacterial systems.

Interleukin-1 Assembles a Proangiogenic Signaling Module Consisting of Caveolin-1, Tumor Necrosis Factor Receptor–Associated Factor 6, p38–Mitogen-Activated Protein Kinase (MAPK), and MAPK-Activated Protein Kinase 2 in Endothelial Cells

Interleukin-1β (IL-1β) is a major cytokine linking inflammation and angiogenesis in pathological vascular processes, such as atherosclerosis and tumor neoangiogenesis. However, signaling pathways mediating IL-1β-induced proangiogenic processes in endothelial cells (ECs) have barely been elucidated yet. Therefore, the present study investigated IL-1β-induced proangiogenic signaling in ECs. IL-1β potently induced tube formation and migration of ECs. This was associated with and dependent on activation of p38-mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) as determined by pharmacological inhibition and gene silencing. Furthermore, silencing of the adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) (lentiviral short hairpin RNA) inhibited these IL-1β-induced processes. Moreover, IL-1β promoted translocation of TRAF6 to insoluble cellular fractions (containing membrane rafts/caveolae) and interaction of TRAF6 with caveolin-1. Accordingly, cellular cholesterol depletion (cyclodextrin) and silencing of caveolin-1 (small interfering RNA) inhibited IL-1β-induced activation of p38-MAPK and MK2, as well as IL-1β-induced tube formation and migration. Finally, silencing of TRAF6 and MK2 deficiency inhibited IL-1β-induced microvessel outgrowth in murine aortic rings ex vivo, and deficiency of MK2 or caveolin-1 significantly reduced IL-1β-induced angiogenesis in mice in vivo (Matrigel plug assay). IL-1β assembles a proangiogenic signaling module consisting of caveolin-1, TRAF6, p38-MAPK, and MK2 in ECs, representing a potential target to intervene into angiogenesis-dependent processes and diseases.

Development of theca specific antisera for the profiling of cell surface proteins in the marine toxic dinoflagellate genus Alexandrium Halim

Revealing the profile of dinoflagellate cell surface proteins (CSPs) is a crucial step for developing molecular probes for effective identification, separation and enumeration of toxic and non-toxic dinoflagellates. This study aimed to develop theca-specific antibodies against the dinoflagellates Alexandrium affine (non-toxic) and A. tamarense (toxic) to distinguish the two species, and verify if these antibodies would enable the analysis of multiple CSPs for probing phytoplankton's nutrient physiology, and facilitating rapid detection and enumeration of these harmful algal species. Using 2-DE immunoblots, we evaluated the specificity and effectiveness of the theca-specific polyclonal antisera against two types of antigens generated from fresh or fixed whole cell and insoluble cellular fractions, respectively. Our results showed that, of the four cell surface antigens, paraformaldehyde fixed whole cell antigen derived antiserum specifically recognized weakly bound theca-associated CSPs in toxic Alexandrium strain. Using the optimized theca-specific antisera, about 187 and 110 cell surface associated antigenic spots were identified on the 2-DE immunoblots of A. affine and A. tamarense, respectively. This immunoproteomic approach is proven to be very useful for phytoplankton CSP studies, permitting a more in-depth elucidation of the relationship among nutrient condition, bloom dynamic and toxin production of the harmful algae in the marine environment.

Inhibition of Cytokine Signaling through Activation of Jak2 Ubiquitination by WP1130.

Abstract 2907Poster Board II-883Constitutive activation of Jak2 through mutations or cytokine stimulation leads to abnormal expansion of the myeloid lineage and plays a key role in multiple myeloproliferative disorders. Kinase-specific inhibitors that prevent substrate phosphorylation can provide effective therapy for these disorders. However, this approach is sometimes inadequate as additional mutations and target-specific modifications can alter the kinase in a sufficient fashion to reduce kinase inhibitor affinity. Therefore additional approaches and molecules may be needed to reduce kinase activity through alternate mechanisms. In this report we describe a novel small molecule-mediated approach to block the Jak2/Stat signaling pathway. Using a previously identified small molecule with Jak2 inhibitory activity, we performed structure-activity relationship studies of AG490 derivatives in a cell-based analysis for inhibition of cytokine-stimulated Stat activation. This effort lead to the synthesis of WP1130 which suppressed Jak2 driven Stat activation with nM to μM potency. However, WP1130 did not act as a direct Jak2 kinase inhibitor. Rather, WP1130 treatment induced a rapid, time and dose-dependent trafficking of Jak2 from the detergent soluble to insoluble cellular fraction. We observed a robust induction of protein ubiquitination in WP1130 treated cells which was not directly or indirectly due to proteosome inhibition. Further, WP1130-mediated ubiquitination was unique with respect to other compounds as it did not reduce association of Jak2 with chaperones, did not induce ER stress or increase reactive oxygen species to activate protein ubiquitination. Jak2 was poly-ubiquitinated, primarily with lysine63-linked polymers, after WP1130 treatment and ubiquitination correlated with Jak2 mobilization without its proteolysis. Sub-cellular fractionation and analysis using confocal microscopy suggested rapid trafficking of Jak2 into cellular structures called aggresomes, in complex with Hsp90, Hsp70 and HDAC6. Lysates from WP1130 treated cells had diminished capacity to hydrolyze ubiquitinated substrates, suggesting the involved of reduced deubiquitinase activity as a mediator of Jak2 ubiquitination in WP1130-treated cells. Together, these results suggest that WP1130 enhances a resident ubiquitination cascade that results in Jak2 ubiquitination and its displacement from its peri-membranous, signaling competent sites. The results also suggest that Jak2 is able to undergo K63-linked poly-ubiquitination and may be exploitable as an alternate approach to interrupt Jak/Stat signaling. Disclosures:No relevant conflicts of interest to declare.

RanBP10 Is a Cytoplasmic Guanine Nucleotide Exchange Factor That Modulates Noncentrosomal Microtubules

Microtubule spindle assembly in mitosis is stimulated by Ran.GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including megakaryocytes. In these cells, platelet biogenesis requires massive mobilization of microtubules in the cell periphery, where they form proplatelets, the immediate precursors of platelets, in the apparent absence of centrioles. Here we identify a cytoplasmic Ran-binding protein, RanBP10, as a factor that binds beta-tubulin and associates with megakaryocyte microtubules. Unexpectedly, RanBP10 harbors GEF activity toward Ran. A point mutation in the candidate GEF domain abolishes exchange activity, and our results implicate RanBP10 as a localized cytoplasmic Ran-GEF. RNA interference-mediated loss of RanBP10 in cultured megakaryocytes disrupts microtubule organization. These results lead us to propose that spatiotemporally restricted generation of cytoplasmic Ran.GTP may influence organization of the specialized microtubules required in thrombopoiesis and that RanBP10 might serve as a molecular link between Ran and noncentrosomal microtubules.

Degrasyn-Induced Trafficking of BCR-ABL as a Novel Mechanism of Kinase Inactivation.

Inhibitors that inactivate specific tyrosine kinases have proven to be a very effective form of therapy of many leukemias and hematopoetic disorders. Most inhibitors function by competing for the ATP-binding pocket or by preventing association with protein substrates. However, clinical and molecular studies have shown that small changes in the structure of the target kinase (point mutations, post-translational modification) affect inhibitor binding affinities, resulting in resistance to this class of inhibitor. Therefore, development of agents that reduce the activity of leukemogenic kinases through alternate mechanisms are of great interest. We previously described a novel class of compounds, termed degrasyns, which reduced BCR-ABL (and Jak2) kinase activity through a unique mechanism. Degrasyn treatment of CML or BaF3 cells expressing wild-type or mutant (T315I) BCR-ABL resulted in a reduction of cytoplasmic BCR-ABL protein levels and loss of downstream signaling without a direct effect on BCR-ABL enzymatic activity. Biochemical studies demonstrated that degrasyn induced a rapid translocation of BCR-ABL from the cytosol to the cytoskeletal fraction (complete within 60 min) and this event correlated with loss of BCR-ABL signaling and initiation of apoptosis. A GFP-fusion protein composed of wild-type or T315I mutant BCR-ABL demonstrated that degrasyn induced high density translocation to the cytoskeletal fraction, as determined by direct fluorescence imaging. Translocation was specific for BCR-ABL and not other kinases (except Jak2) or signaling proteins. Translocation correlated with degrasyn-mediated tyrosine phosphorylation of a subset of specific proteins, including the tyrosine kinase Lyn, in the insoluble cellular fraction. CML cells expressing high levels of Lyn were more sensitive to degrasyn-mediated apoptosis suggesting that Lyn plays a role in degrasyn activity. Together these results suggest that degrasyn inactivates BCR-ABL by inducing its translocation to a cellular compartment that prevents its participation in oncogenic signaling. Degrasyn-induced BCR-ABL translocation was not effected by mutations that block dasatinib and imatinib activity and shows greater activity against some forms of imatinib resistance (Lyn overexpression). Animal studies demonstrated that degrasyn has anti-leukemic activity and functions through a unique mechanism of action. Development of inhibitors with this mode of action may be of significance for CML patients that fail therapy with ATP-binding pocket-directed tyrosine kinase inhibitors.

The inverse agonist activity of soluble CTLA-4 ligands requires oligomerization of this receptor and CD28 expression (88.26)

Abstract CTLA-4 can inhibit or activate T cells depending on the ligand it engages. We have shown that CTLA-4 ligation with a bi-specific in-tandem single chain Fv, known as 24:26, can activate CTLA-4-expressing primary human T cells in the absence of TCR and CD28 engagement. To understand the basis of such an inverse agonist activity, we examined the requirements for CTLA-4-mediated T cell activation. We observed that 24:26 and soluble B7.1 Ig, but not B7 molecules on APCs, induced the formation of a unique CTLA-4 oligomer that disappeared from the soluble cell lysate fraction and subsequently appeared in the insoluble cellular fraction. Oligomerization occurred upon ligation of CTLA-4 homodimers and was necessary but not sufficient for T cell activation. The inverse agonist activity of these soluble ligands required the cytoplasmic domain of CTLA-4 since CTLA-4 molecules lacking this domain did not induce T cell activation. CD28 expression was necessary for the inhibitory and activating functions of CTLA-4 suggesting a functional interaction between these two receptors. Understanding the functional responses to soluble ligands of costimulatory receptors is important in the wake of the side effects observed with CD28 superagonists. Our data indicates that the biological effect of soluble ligands of CTLA-4 will be determined by the oligomerization of this receptor. Supported by the Canadian Institutes of Health Research.

Expression and Targeting of CX3CL1 (Fractalkine) in Renal Tubular Epithelial Cells

The chemokine CX3CL1 plays a key role in glomerulonephritis and can act as both chemoattractant and adhesion molecule. CX3CL1 also is upregulated in tubulointerstitial injury, but little is known about the subcellular distribution and function of CX3CL1 in renal tubular epithelial cells (RTEC). Unexpectedly, it was found that CX3CL1 is expressed predominantly on the apical surface of tubular epithelium in human renal transplant biopsy specimens with acute rejection or acute tubular necrosis. For studying the targeting of CX3CL1 in polarized RTEC, MDCK cells that expressed untagged or green fluorescent protein-tagged CX3CL1 were generated. The chemokine was present on the apical membrane and in subapical vesicles. Apical targeting of CX3CL1 was not due to signals that were conferred by its intracellular domain, to associations with lipid rafts, or to O-glycosylation but, rather, depended on N-linked glycosylation of the protein. With the use of fluorescence recovery after photobleaching, it was found that CX3CL1 is immobile in the apical membrane. However, CX3CL1 partitioned with the triton-soluble rather than -insoluble cellular fraction, indicating that it is not associated directly with the actin cytoskeleton or with lipid rafts. Accordingly, disruption of rafts through cholesterol depletion did not render CX3CL1 mobile. For exploration of potential functions of apical CX3CL1, binding of CX3CR1-expressing leukocytes to polarized RTEC was examined. Leukocyte adhesion to the luminal surface was enhanced significantly when CX3CL1 was present. These data demonstrate that CX3CL1 is expressed preferentially on the apical membrane of RTEC and suggest a novel function for the chemokine in recruitment and retention of leukocytes in tubulointerstitial inflammation.

Anthrax Toxin Receptor 1/Tumor Endothelium Marker 8 Mediates Cell Spreading by Coupling Extracellular Ligands to the Actin Cytoskeleton

Tumor endothelial marker 8 (TEM8) is induced in tumor-associated vasculature and acts as a receptor for Protective Antigen (PA), the cell-binding component of the anthrax toxin determinant for toxin entrance into cells. However, the normal function for TEM8 remains unknown. We show that TEM8 functions as an adhesion molecule mediating cell spreading on immobilized PA and collagen I. The mechanism for TEM8 interaction with collagen I was cell type-specific, because binding to collagen I was abrogated by beta1 integrin function blocking antibody in HEK293 cells, but not in primary synovial rabbit fibroblasts. Binding to PA remained unaffected by the addition of beta1 integrin function blocking antibody. Whereas the extracellular and transmembrane domains of TEM8 were sufficient to provide cell attachment, the intracellular domain was critical for spreading. Fusion of the cytosolic domain of TEM8 to the IL-2 receptor, conferred cell-spreading capability on IL-2 receptor antibody substrates. The cytoplasmic domain mediated linkage with the actin cytoskeleton as it co-precipitated actin and determined partitioning of TEM8 to the actin-containing detergent insoluble cellular fraction. TEM8 anchorage to actin was relevant as spreading was inhibited by the cytoskeleton-disrupting drug cytochalasin D, but persisted in the presence of the microtubule-depolymerizing drug nocodazole, and in cells lacking intermediate filaments. Thus, our results indicate that TEM8 is a new adhesion molecule linking collagen I or PA to the actin cytoskeleton.

Peptide Aptamer-mediated Inhibition of Target Proteins by Sequestration into Aggresomes

Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.

Melatonin increases survival of HaCaT keratinocytes by suppressing UV‐induced apoptosis

Melatonin is a potent antioxidant and direct radical scavenger. As keratinocytes represent the major population in the skin and UV light causes damage to these cells, the possible protective effects of melatonin against UV-induced cell damage in HaCaT keratinocytes were investigated in vitro. Cells were preincubated with melatonin at graded concentrations from 10(-9) to 10(-3) m for 30 min prior to UV irradiation at doses of 25 and 50 mJ/cm2. Biological markers of cellular viability such as DNA synthesis and colony-forming efficiency as well as molecular markers of apoptosis were measured. DNA synthesis was determined by [3H]-thymidine incorporation into insoluble cellular fraction, clonogenicity through plating efficiency experiments and apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. DNA synthesis experiments showed a strong protective effect by preincubation with melatonin at concentrations of 10(-4) m (P < 0.01) and 10(-3) m (P < 0.001). Additional postirradiation treatment with melatonin showed no increase in the pre-UV incubation protective effect. These results indicate that preincubation is a requirement for melatonin to exert its protective effects. The mechanism of melatonin's protective effect (10(-6) to 10(-3) m) includes inhibition of apoptosis as measured by TUNEL assay. Moreover, the biological significance of these effects is supported by clonogenic studies showing a significantly higher number of colonies in cultures treated with melatonin compared to controls. Thus, pretreatment with melatonin led to strong protection against UVB-induced damage in keratinocytes.