HSC70 as a sensor of low temperature: role in cold-triggered autoinflammatory disorders.

Familial cold autoinflammatory syndrome (FCAS) is a subset of heritable autoinflammatory disorders wherein inflammatory symptoms aggravate upon exposure of the individual to subnormal temperature. In the past two decades, several mutations in various genes such as NLRP3, NLRP12, PLCG2 and NLRC4 have been identified that cause cold-triggered inflammation. However, our understanding of the mechanisms by which cells perceive subnormal temperature, and what keeps the inflammation under check until exposure to low temperature, is very limited. We hypothesise that recognition of FCAS-associated mutants as misfolded polypeptides by temperature-sensitive HSC70 (HSPA8) chaperone determines the FCAS phenotype. At 37 °C, HSC70 would interact with the mutant proteins, keeping them almost inactive, and loss of interaction at low temperature due to a conformational change in HSC70 would lead to their activation. The proposed mechanism of low temperature sensing in the context of FCAS may have wider implications for HSC70 as a cold temperature sensor in various pathological conditions where symptoms get aggravated upon exposure to low temperature.

Human primary retinal cells as an in-vitro model for investigating defective signalling caused by OPTN mutants associated with glaucoma.

Studies carried out on the pathogenesis of glaucoma using murine cell lines and animal models require to be validated in human cells. Therefore, we explored the possibility of using human primary retinal cells (hPRCs) in culture as a model for molecular studies and testing of potential therapeutic drugs. For this purpose, central retinal tissue, obtained from the enucleated eyes of patients with anterior staphyloma, was digested with trypsin and grown in a medium containing supplements (basic fibroblast growth factor and fetal bovine serum). hPRCs at passage 1 and 2, show expression of either GFAP, a glial cell marker, or ß-III tubulin, a retinal ganglion cell (RGC)-specific marker. But at passages 3-5 nearly all of hPRCs express several RGC-specific markers (Brn3 proteins, Thy-1, ß-III tubulin, RBPMS and NeuN) but not GFAP. Expression of these markers indicated that these cells may have functional properties of RGCs. As RGCs are sensitive to glaucoma-associated mutants of OPTN, we analysed the survival of hPRCs upon overexpression of OPTN mutants. Glaucoma-associated mutants, E50K-OPTN and M98K-OPTN, induced significantly higher cell death in hPRCs compared to WT-OPTN, whereas an amyotrophic lateral sclerosis-associated mutant, E478G-OPTN, did not. TBK1 inhibitor Amlexanox protected hPRCs from E50K-OPTN and M98K-OPTN induced cell death. M98K-OPTN induced cell death was suppressed by inhibitors of CaMKKß and AMPK in hPRCs as well as in 661W, a mouse cell line that expresses several markers of RGCs and RGC precursor cells. Our results suggest that hPRCs under appropriate culture condition show RGC-like properties. These cells can be used to explore the molecular mechanisms of cell death relevant for glaucoma pathogenesis and for testing of cytoprotective compounds.

C3G Regulates STAT3, ERK, Adhesion Signaling, and Is Essential for Differentiation of Embryonic Stem Cells.

C3G (RAPGEF1), engaged in multiple signaling pathways, is essential for the early development of the mouse. In this study, we have examined its role in mouse embryonic stem cell self-renewal and differentiation. C3G null cells generated by CRISPR mediated knock-in of a targeting vector exhibited enhanced clonogenicity and long-term self-renewal. They did not differentiate in response to LIF withdrawal when compared to the wild type ES cells and were defective for lineage commitment upon teratoma formation in vivo. Gene expression analysis of C3G KO cells showed misregulated expression of a large number of genes compared with WT cells. They express higher levels of self-renewal factors like KLF4 and ESRRB and show high STAT3 activity, and very low ERK activity compared to WT cells. Reintroduction of C3G expression in a KO line partially reverted expression of ESRRB, and KLF4, and ERK activity similar to that seen in WT cells. The expression of self-renewal factors was persistent for a longer time, and induction of lineage-specific markers was not seen when C3G KO cells were induced to form embryoid bodies. C3G KO cells showed poor adhesion and significantly reduced levels of pFAK, pPaxillin, and Integrin-ß1, in addition to downregulation of the cluster of genes involved in cell adhesion, compared to WT cells. Our results show that C3G is essential for the regulation of STAT3, ERK, and adhesion signaling, to maintain pluripotency of mouse embryonic stem cells and enable their lineage commitment for differentiation.

Complex formation and reciprocal regulation between GSK3ß and C3G.

GSK3ß, a ubiquitously expressed Ser/Thr kinase, regulates cell metabolism, proliferation and differentiation. Its activity is spatially and temporally regulated dependent on external stimuli and interacting partners, and its deregulation is associated with various human disorders. In this study, we identify C3G (RapGEF1), a protein essential for mammalian embryonic development as an interacting partner and substrate of GSK3ß. In vivo and in vitro interaction assays demonstrated that GSK3ß and Akt are present in complex with C3G. Molecular modelling and mutational analysis identified a domain in C3G that aids interaction with GSK3ß, and overlaps with its nuclear export sequence. GSK3ß phosphorylates C3G on primed as well as unprimed sites, and regulates its subcellular localization. Over-expression of C3G resulted in activation of Akt and inactivation of GSK3ß. Huntingtin aggregate formation, dependent on GSK3ß inhibition, was enhanced upon C3G overexpression. Stable clones of C2C12 cells generated by CRISPR/Cas9 mediated knockdown of C3G, that cannot differentiate, show reduced Akt activity and S9-GSK3ß phosphorylation compared to wild type cells. Co-expression of catalytically active GSK3ß inhibited C3G induced myocyte differentiation. C3G mutant defective for GSK3ß phosphorylation, does not alter S9-GSK3ß phosphorylation and, is compromised for inducing myocyte differentiation. Our results show complex formation and reciprocal regulation between GSK3ß and C3G. We have identified a novel function of C3G as a negative regulator of GSK3ß, a property important for its ability to induce myogenic differentiation.

Expression of a novel brain specific isoform of C3G is regulated during development.

Mice lacking C3G (RapGEF1), a ubiquitously expressed protein essential for neuronal differentiation, show multiple defects in brain development. Function of C3G in neurogenesis is poorly defined. Here, we identify brain specific expression of a novel C3G isoform in mice and humans. This isoform has an insert in the Crk-binding region, generating a polypeptide of 175 kDa, unlike the previously known 140 kDa form expressed in all other tissues. In the adult mouse brain, C3G expression is seen in neurons, but was not detectable in GFAP-positive cells. C3G levels were high in the CA3 region of hippocampus and in mitral cells of olfactory bulb. Neural progenitor cells positive for Doublecortin and Nestin, show expression of C3G. During development, C3G is expressed in precursor cells prior to their differentiation into mature neurons or astrocytes. The 175 kDa as well as 140 kDa forms are seen in embryonic mouse brain, while only the 175 kDa variant is seen in post-natal brain. Human cerebral organoids generated from induced pluripotent stem cells predominantly expressed the 140 kDa polypeptides, and the 175 kDa isoform appeared upon maturation. This study describes developmental regulation and neuronal expression of a brain specific isoform of C3G, a molecule essential for normal development of the mammalian brain.

C3G localizes to the mother centriole in a cenexin-dependent manner and regulates centrosome duplication and primary cilium length.

C3G (also known as RAPGEF1) plays a role in cell differentiation and is essential for early embryonic development in mice. In this study, we identify C3G as a centrosomal protein that colocalizes with cenexin (also known as ODF2) at the mother centriole in interphase cells. C3G interacts with cenexin through its catalytic domain, and the two proteins show interdependence for localization to the centrosome. C3G depletion causes a decrease in cellular cenexin levels. Centrosomal localization of C3G is lost as myocytes differentiate to form myotubes. Depletion of C3G by CRISPR/Cas9 results in the formation of supernumerary centrioles, whereas overexpression of C3G, or expression of a catalytically active C3G deletion construct, inhibits centrosome duplication. Cilium length is increased in C3G knockout cells, and this phenotype is reverted upon reintroduction of C3G or its catalytic domain alone. Association of C3G with the basal body is dynamic, decreasing upon serum starvation and increasing upon re-entry into the cell cycle. C3G inhibits cilium formation and length, and this inhibition is dependent on C3G catalytic activity. We conclude that C3G regulates centrosome duplication and maintains ciliary homeostasis, properties that could be important for its role in embryonic development.

The need to develop a framework for human-relevant research in India: Towards better disease models and drug discovery.

The low translational efficiency of animal models to humans, and the development of new-age methodologies that are human-cell based, is fuelling a paradigm change across the globe. In this perspectives paper, we discuss the current state of research, funding, and regulation in these 21st century technologies, including organoids and organ-on-chip in India. Recently, a road-map was drawn by Indian Council for Medical Research (ICMR) regarding alternatives to animals in research in India and it also held a special session in January 2018 to discuss latest developments in new human-relevant model systems. We document the regulatory and research landscape in this field in India. We also discuss the challenges present in this field which include lack of training and skills to handle embryonic or induced pluripotent stem cell (iPSC) lines, funding limitations, lack of domestic production of reagents leading to elevated costs, and lack of infrastructure, such as microfabrication facilities. In the end, we provide recommendations to enable innovation and application of human-relevant methodologies to develop India as a key player in this arena globally.

HSC70 regulates cold-induced caspase-1 hyperactivation by an autoinflammation-causing mutant of cytoplasmic immune receptor NLRC4.

NLRC4 [nucleotide-binding domain and leucine-rich repeat (NLR) family, caspase recruitment domain (CARD) containing 4] is an innate immune receptor, which, upon detection of certain pathogens or internal distress signals, initiates caspase-1-mediated interleukin-1ß maturation and an inflammatory response. A gain-of-function mutation, H443P in NLRC4, causes familial cold autoinflammatory syndrome (FCAS) characterized by cold-induced hyperactivation of caspase-1, enhanced interleukin-1ß maturation, and inflammation. Although the H443P mutant shows constitutive activity, the mechanism involved in hyperactivation of caspase-1 by NLRC4-H443P upon exposure of cells to lower temperature is not known. Here, we show that heat shock cognate protein 70 (HSC70) complexes with NLRC4 and negatively regulates caspase-1 activation by NLRC4-H443P in human cells. Compared with NLRC4, the structurally altered NLRC4-H443P shows enhanced interaction with HSC70. Nucleotide binding- and leucine-rich repeat domains of NLRC4, but not its CARD, can engage in complex formation with HSC70. Knockdown of HSC70 enhances apoptosis-associated speck-like protein containing a CARD (ASC)-speck formation and caspase-1 activation by NLRC4-H443P. Exposure to subnormal temperature results in reduced interaction of NLRC4-H443P with HSC70, and an increase in its ability to form ASC specks and activate caspase-1. Unlike the NLRC4-H443P mutant, another constitutively active mutant (NLRC4-V341A) associated with autoinflammatory diseases, but not FCAS, showed neither enhanced interaction with HSC70 nor an increase in inflammasome formation upon exposure to subnormal temperature. Our results identify HSC70 as a negative regulator of caspase-1 activation by the temperature-sensitive NLRC4-H443P mutant. We also show that low-temperature-induced hyperactivation of caspase-1 by NLRC4-H443P is due to loss of inhibition by HSC70.

Development and characterization of a novel monoclonal antibody that recognizes an epitope in the central protein interaction domain of RapGEF1 (C3G).

The ubiquitously expressed protein RapGEF1 (C3G) regulates multiple cellular activities and is essential for early embryonic development in mammals. It has functions dependent on its catalytic activity as well as protein interaction domain and regulates ß-catenin signaling. This study describes the generation of a novel monoclonal antibody, 3F6mAb and its characterization for recognition of RapGEF1. Mice were immunized with recombinant protein having only the Crk binding region of RapGEF1 and hybridoma clones created by fusion of immunized spleen cells with Sp2/0 myeloma cells. This antibody recognizes human, primate and murine RapGEF1 protein. Based on the recognition of various deletion constructs, we have mapped its epitope to 580-648 amino acids. Isotyping showed that it belongs to IgG1 class of heavy chain and Kappa light chain. 3F6mAb is suitable for detecting cellular RapGEF1 by western-blotting, immunofluorescence and immunoprecipitation. It has an advantage over most of the commercially available antibodies as it can detect N- and C-terminal truncated forms of RapGEF1. Using this antibody to detect mobility shift, we show that RapGEF1 is phosphorylated on tyrosine as well as S/T residues in its Crk binding domain. This monoclonal antibody is a valuable tool that will aid in understanding functions of cellular RapGEF1.

C3G dynamically associates with nuclear speckles and regulates mRNA splicing.

C3G (Crk SH3 domain binding guanine nucleotide releasing factor) (Rap guanine nucleotide exchange factor 1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleocytoplasmic exchange. Here we show that C3G localizes to SC35-positive nuclear speckles and regulates splicing activity. Reversible association of C3G with speckles was seen on inhibition of transcription and splicing. C3G shows partial colocalization with SC35 and is recruited to a chromatin and RNase-sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton and is lost on expression of the kinase Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles, and inactivation of Rap signaling by expression of GFP-Rap1GAP alters speckle morphology and number. Enhanced association of C3G with speckles is seen on glycogen synthase kinase 3 beta inhibition or differentiation of C2C12 cells to myotubes. CRISPR/Cas9-mediated knockdown of C3G resulted in altered splicing activity of an artificial gene as well as endogenous CD44. C3G knockout clones of C2C12 as well as MDA-MB-231 cells showed reduced protein levels of several splicing factors compared with control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.

661W is a retinal ganglion precursor-like cell line in which glaucoma-associated optineurin mutants induce cell death selectively.

A photoreceptor cell line, 661W, derived from a mouse retinal tumor that expresses several markers of cone photoreceptor cells has been described earlier. However, these cells can be differentiated into neuronal cells. Here, we report that this cell line expressed certain markers specific to retinal ganglion cells such as Rbpms, Brn3b (Pou4f2), Brn3c (Pou4f3), Thy1 and γ-synuclein (Sncg), and some other markers of neuronal cells (beta-III tubulin, NeuN and MAP2). These cells also expressed Opn1mw, a cone-specific marker and nestin, a marker for neural precursor cells. Two glaucoma-associated mutants of OPTN, E50K and M98K, but not an amyotrophic lateral sclerosis-associated mutant, E478G, induced cell death selectively in 661W cells. However, in a motor neuron cell line, NSC34, E478G mutant of OPTN but not E50K and M98K induced cell death. We conclude that 661W is a retinal ganglion precursor-like cell line, which shows properties of both retinal ganglion and photoreceptor cells. We suggest that these cells could be utilized for exploring the mechanisms of cell death induction and cytoprotection relevant for glaucoma pathogenesis. RGC-5 cell line which probably arose from 661W cells showed expression of essentially the same markers of retinal ganglion cells and neuronal cells as seen in 661W cells.

C3G shows regulated nucleocytoplasmic exchange and represses histone modifications associated with euchromatin.

C3G (RapGEF1) is a ubiquitously expressed guanine nucleotide exchange factor that functions in signaling pathways regulating cell proliferation, apoptosis, and actin reorganization. It is essential for differentiation and early embryonic development in mice. Overexpressed C3G shows predominant cytoplasmic localization, but endogenous C3G is a component of nuclear fractions in a variety of cell types. Coexpression of importin-α and inhibition of nuclear export by leptomycin B resulted in predominant nuclear localization of C3G. Functional NLSs, NES, and GSK3-ß-dependent phosphorylation regulate its dynamic nuclear localization. C3G translocates to the nucleus in response to myogenic differentiation and sublethal dose of cisplatin. C3G is associated with chromatin and nuclear matrix fractions. Cells with C3G localized in the nucleus showed peripheralization of heterochromatin and reduced histone modifications associated with euchromatin. Short hairpin RNA-mediated depletion of C3G in epithelial cells resulted in reduced expression of CDK inhibitors and the histone demethylase KDM5A. Myoblast clones with CRISPR/Cas9-mediated knockout of C3G failed to show repression of histone marks and did not show up-regulation of myosin heavy chain and myotube formation when grown in differentiation medium. Our results document regulated nucleocytoplasmic exchange of C3G in response to physiological stimuli and provide insights into nuclear functions for C3G.

CDC20siRNA and paclitaxel co-loaded nanometric liposomes of a nipecotic acid-derived cationic amphiphile inhibit xenografted neuroblastoma.

Despite significant recent progress in the area of translational genomics of neuroblastoma, the overall survival rates for children with high-risk NB continue to be not more than 5 years due to tumor relapse and/or drug-resistant tumors. Herein we report on the development of a neuroblastoma targeting nanometric (130-150 nm) circulation stable liposomal system prepared from a novel nipecotic acid-derived cationic amphiphile (NACA). The size ranges of liposomes (130-150 nm) were confirmed by both dynamic light scattering and transmission electron microscopy. The findings in the gel electrophoresis assay revealed that siRNAs encapsulated within the liposomes of NACA (with 90% entrapment efficiency) are protected from attack by RNase. Cellular uptake experiments using FAM-siRNA loaded liposomes of NACA showed the liposomal entry in human neuroblastoma cells (IMR-32) to be mediated via the GABAA receptor. CDC20siRNA-loaded liposomes of NACA caused significantly higher CDC20 gene silencing efficiency in IMR-32 cells compared to CDC20 gene knockdown efficiency mediated by CDC20siRNA-loaded control non-targeting liposomes (NTL). The findings in the annexin-V binding based flow cytometric apoptosis assay and MTT-based cellular cytotoxicity assay support the notion that pronounced (80%) neuroblastoma cell death upon treatment with CDC20siRNA & PTX co-loaded liposomes of NACA presumably originates from enhanced apoptosis of cells. Importantly, intravenously administered CDC20siRNA & PTX co-loaded liposomes of NACA significantly inhibited growth of xenografted human neuroblastoma in athymic nude mice. The presently disclosed strategy of co-delivering potent anticancer siRNA and small molecule chemotherapeutics using liposomes of NACA opens a new door for combating the dreaded disease of neuroblastoma.

A Disease-associated Mutant of NLRC4 Shows Enhanced Interaction with SUG1 Leading to Constitutive FADD-dependent Caspase-8 Activation and Cell Death.

Nod-like receptor family card containing 4 (NLRC4)/Ipaf is involved in recognition of pathogen-associated molecular patterns leading to caspase-1 activation and cytokine release, which mediate protective innate immune response. Point mutations in NLRC4 cause autoinflammatory syndromes. Although all the mutations result in constitutive caspase-1 activation, their phenotypic presentations are different, implying that these mutations cause different alterations in properties of NLRC4. NLRC4 interacts with SUG1 and induces caspase-8-mediated cell death. Here, we show that one of the autoinflammatory syndrome-causing mutants of NLRC4, H443P, but not T337A and V341A, constitutively activates caspase-8 and induces apoptotic cell death in human lung epithelial cells. Compared with wild type NLRC4, the H443P mutant shows stronger interaction with SUG1 and with ubiquitinated cellular proteins. Phosphorylation of NLRC4 at Ser533 plays a crucial role in caspase-8 activation and cell death. However, H443P mutant does not require Ser533 phosphorylation for caspase-8 activation and cell death. Caspase-8 activation by NLRC4 and its H443P mutant are dependent on the adaptor protein FADD. A phosphomimicking mutant of NLRC4, S533D does not require SUG1 activity for inducing cell death. Ubiquitin-tagged NLRC4 could induce cell death and activate caspase-8 independent of Ser533 phosphorylation. Our work suggests that SUG1-mediated signaling results in enhanced ubiquitination and regulates FADD-dependent caspase-8 activation by NLRC4. We show that the autoinflammation-associated H443P mutant is altered in interaction with SUG1 and ubiquitinated proteins, triggering constitutive caspase-8-mediated cell death dependent on FADD but independent of Ser533 phosphorylation.

Use of Dominant-Negative/Substrate Trapping PTP Mutations to Search for PTP Interactors/Substrates.

Phosphorylation of proteins on tyrosine residues is the consequence of coordinated action of tyrosine kinases (TKs), and protein tyrosine phosphatases (PTPs). Together, they regulate intermolecular interactions, subcellular localization, and activity of a variety of proteins. The level of total protein-associated tyrosine phosphorylation in eukaryotic cells is only a small fraction of the total phosphorylation. PTPs, which have high specific activity compared to tyrosine kinases, play an important role in maintaining the tyrosine phosphorylation state of proteins and regulate signal transduction pathways and cellular responses. PTPs depend on specific invariant residues that enable binding to substrates phosphorylated at tyrosine and aid catalytic activity. Identification of PTP substrates has helped understand their role in distinct intracellular signaling pathways. Because of their high specific activity, the interaction between tyrosine phosphatases and their substrates is often very transient in the cellular context, and therefore identification of physiological substrates has been difficult. Single-site mutations in the enzymes stabilize interaction between the enzyme and its targets and have been used extensively to identify substrates. The mutations are either of the catalytic cysteine (Cys) residue or other invariant residues and have been classified as substrate-trapping mutants (STMs). These mutants often serve as dominant negatives that can inactivate effector functions of a specific PTP within cells. Considering their association with human disorders, inhibiting specific PTPs is important therapeutically. Since the catalytic domains are largely conserved, developing small-molecule inhibitors to a particular enzyme has proven difficult and therefore alternate strategies to block functions of individual enzymes are seriously being investigated. We provide a description of methods that will be useful to design strategies of using dominant-negative and substrate-trapping mutants for identifying novel interacting partners and substrates of PTPs.

A Glaucoma-Associated Variant of Optineurin, M98K, Activates Tbk1 to Enhance Autophagosome Formation and Retinal Cell Death Dependent on Ser177 Phosphorylation of Optineurin.

Certain missense mutations in optineurin/OPTN and amplification of TBK1 are associated with normal tension glaucoma. A glaucoma-associated variant of OPTN, M98K, induces autophagic degradation of transferrin receptor (TFRC) and death in retinal cells. Here, we have explored the role of Tbk1 in M98K-OPTN-induced autophagy and cell death, and the effect of Tbk1 overexpression in retinal cells. Cell death induced by M98K-OPTN was dependent on Tbk1 as seen by the effect of Tbk1 knockdown and blocking of Tbk1 activity by a chemical inhibitor. Inhibition of Tbk1 also restores M98K-OPTN-induced transferrin receptor degradation. M98K-OPTN-induced autophagosome formation, autophagy and cell death were dependent on its phosphorylation at S177 by Tbk1. Knockdown of OPTN reduced starvation-induced autophagosome formation. M98K-OPTN expressing cells showed higher levels of Tbk1 activation and enhanced phosphorylation at Ser177 compared to WT-OPTN expressing cells. M98K-OPTN-induced activation of Tbk1 and its ability to be phosphorylated better by Tbk1 was dependent on ubiquitin binding. Phosphorylated M98K-OPTN localized specifically to autophagosomes and endogenous Tbk1 showed increased localization to autophagosomes in M98K-OPTN expressing cells. Overexpression of Tbk1 induced cell death and caspase-3 activation that were dependent on its catalytic activity. Tbk1-induced cell death possibly involves autophagy, as shown by the effect of Atg5 knockdown, and requirement of autophagic function of OPTN. Our results show that phosphorylation of Ser177 plays a crucial role in M98K-OPTN-induced autophagosome formation, autophagy flux and retinal cell death. In addition, we provide evidence for cross talk between two glaucoma associated proteins and their inter-dependence to mediate autophagy-dependent cell death.

E50K-OPTN-induced retinal cell death involves the Rab GTPase-activating protein, TBC1D17 mediated block in autophagy.

The protein optineurin coded by OPTN gene is involved in several functions including regulation of endocytic trafficking, autophagy and signal transduction. Certain missense mutations in the gene OPTN cause normal tension glaucoma. A glaucoma-causing mutant of optineurin, E50K, induces death selectively in retinal cells. This mutant induces defective endocytic recycling of transferrin receptor by causing inactivation of Rab8 mediated by the GTPase-activating protein, TBC1D17. Here, we have explored the mechanism of E50K-induced cell death. E50K-OPTN-induced cell death was inhibited by co-expression of a catalytically inactive mutant of TBC1D17 and also by shRNA mediated knockdown of TBC1D17. Endogenous TBC1D17 colocalized with E50K-OPTN in vesicular structures. Co-expression of transferrin receptor partially protected against E50K-induced cell death. Overexpression of the E50K-OPTN but not WT-OPTN inhibited autophagy flux. Treatment of cells with rapamycin, an inducer of autophagy, reduced E50K-OPTN-induced cell death. An LC3-binding-defective mutant of E50K-OPTN showed reduced cell death, further suggesting the involvement of autophagy. TBC1D17 localized to autophagosomes and inhibited autophagy flux dependent on its catalytic activity. Knockdown of TBC1D17 rescued cells from E50K-mediated inhibition of autophagy flux. Overall, our results suggest that E50K mutant induced death of retinal cells involves impaired autophagy as well as impaired transferrin receptor function. TBC1D17, a GTPase-activating protein for Rab GTPases, plays a crucial role in E50K-induced impaired autophagy and cell death.

M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells.

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.

The tyrosine phosphatase TC48 interacts with and inactivates the oncogenic fusion protein BCR-Abl but not cellular Abl.

The chimeric oncoprotein BCR-Abl exhibits deregulated protein tyrosine kinase activity and is responsible for the pathogenesis of certain human leukemias, such as chronic myelogenous leukemia. The activities of cellular Abl (c-Abl) and BCR-Abl are stringently regulated and the cellular mechanisms involved in their inactivation are poorly understood. Protein tyrosine phosphatases can negatively regulate Abl mediated signaling by dephosphorylating the kinase and/or its substrates. This study investigated the ability of the intracellular T cell protein tyrosine phosphatase (TCPTP/PTPN2) to dephosphorylate and regulate the functions of BCR-Abl and c-Abl. TCPTP is expressed as two alternately spliced isoforms - TC48 and TC45, which differ in their C-termini and localize to the cytoplasm and nucleus respectively. We show that TC48 dephosphorylates BCR-Abl but not c-Abl and inhibits its activity towards its substrate, CrkII. Y1127 and Y1294 residues whose phosphorylation corresponds with BCR-Abl activation status were the primary sites targeted by TC48. Co-localization and immunoprecipitation experiments showed that TC48 interacted with BCR-Abl but not with c-Abl, and BCR domain was sufficient for interaction. TC48 expression resulted in the stabilization of Bcr-Abl protein dependent on its phosphatase activity. Inactivation of cellular TC48 in K562 cells by stable expression of a dominant negative catalytically inactive mutant TC48, enhanced proliferation. TC48 expressing K562 clones showed reduced proliferation and enhanced sensitivity to STI571 compared to control clones suggesting that TC48 can repress the growth of CML cells. This study identifies a novel cellular regulator that specifically inhibits the activity of oncogenic BCR-Abl but not that of the cellular Abl kinase.

Dynamic changes in nuclear localization of a DNA-binding protein tyrosine phosphatase TCPTP in response to DNA damage and replication arrest.

TCPTP is an ubiquitously expressed tyrosine phosphatase with a predominant nuclear isoform (TC45) that binds DNA and has a role in G1-S cell cycle progression. Its deregulation by overexpression induces p53-dependent apoptosis, but the physiological role of its DNA-binding function is not known. Using immunocytochemistry and subcellular fractionation, we investigated changes in its localization in response to DNA damage and replication arrest. Rat fibroblasts showed an increase in endogenous TCPTP bound to nuclear components 3 h after exposure to sublethal dose of UV irradiation. Fractionation of nuclei showed an increase in chromatin and nuclear matrix associated component of TC45. After UV treatment, cells showed a concentration of TCPTP in discrete foci and enhanced colocalization with PCNA and p53BP1. Cells arrested at G1-S transition by hydroxyurea showed a loss of the predominant nuclear staining of TCPTP and an increase in cytoplasmic staining. Upon release from replication block, there was a time-dependent increase in number of cells showing prominent nuclear localization. This change in localization coincides with that of PCNA and Cdk2, two other nuclear proteins having functions in DNA replication. These results provide evidence for the regulation of TCPTP in response to DNA damage and replication stress. Dynamic changes in its localization coincident with that of PCNA suggest involvement of TCPTP in DNA repair and replication.