Pleiotropic functions of TAO kinases and their dysregulation in neurological disorders.

Thousand and one amino acid kinases (TAOKs) are relatively understudied and functionally pleiotropic protein kinases that have emerged as important regulators of neurodevelopment. Through their conserved amino-terminal catalytic domain, TAOKs mediate phosphorylation at serine/threonine residues in their substrates, but it is their divergent regulatory carboxyl-terminal domains that confer both exquisite functional specification and cellular localization. In this Review, we discuss the physiological roles of TAOKs and the intricate signaling pathways, molecular interactions, and cellular behaviors they modulate-from cell stress responses, division, and motility to tissue homeostasis, immunity, and neurodevelopment. These insights are then integrated into an analysis of the known and potential impacts of disease-associated variants of TAOKs, with a focus on neurodevelopmental disorders, pain and addiction, and neurodegenerative diseases. Translating this foundation into clinical benefits for patients will require greater structural and functional differentiation of the TAOKs afforded by their individually specialized domains.

Crystal structure of the pilus-specific sortase from early colonizing oral Streptococcus sanguinis captures an active open-lid conformation.

Dental plaque is a complex microbial biofilm community of many species and a major cause of oral infections and infectious endocarditis. Plaque development begins when primary colonizers attach to oral tissues and undergo coaggregation. Primary colonizers facilitate cellular attachment and inter-bacterial interactions through sortase-dependent pili (or fimbriae) extending out from their cell surface. Consequently, the sortase enzyme is viewed as a potential drug target for controlling biofilm formation and avoiding infection. Streptococcus sanguinis is a primary colonizing bacterium whose pili consist of three different pilin subunits that are assembled together by the pilus-specific (C-type) SsaSrtC sortase. Here, we report on the crystal structure determination of the recombinant wild-type and active-site mutant forms of SsaSrtC. Interestingly, the SsaSrtC structure exhibits an open-lid conformation, although a conserved DPX motif is lacking in the lid. Based on molecular docking and structural analysis, we identified the substrate-binding residues essential for pilin recognition and pilus assembly. We also demonstrated that while recombinant SsaSrtC is enzymatically active toward the five-residue LPNTG sorting motif peptide of the pilins, this activity is significantly reduced by the presence of zinc. We further showed that rutin and α-crocin are potential candidate inhibitors of the SsaSrtC sortase via structure-based virtual screening and inhibition assays. The structural knowledge gained from our study will provide the means to develop new approaches that target pilus-mediated attachment, thereby preventing oral biofilm growth and infection.

Neurodevelopmental disorder-associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase.

Mutations in TAOK1, which encodes a serine-threonine kinase, are associated with both autism spectrum disorder (ASD) and neurodevelopmental delay (NDD). Here, we investigated the molecular function of this evolutionarily conserved kinase and the mechanisms through which TAOK1 mutations may lead to neuropathology. We found that TAOK1 was abundant in neurons in the mammalian brain and remodeled the neuronal plasma membrane through direct association with phosphoinositides. Our characterization of four NDD-associated TAOK1 mutations revealed that these mutants were catalytically inactive and were aberrantly trapped in a membrane-bound state, which induced abnormal membrane protrusions. Expression of these TAOK1 mutants in cultured mouse hippocampal neurons led to abnormal growth of the dendritic arbor. The coiled-coil region carboxyl-terminal to the kinase domain was predicted to fold into a triple helix, and this region directly bound phospholipids and was required for both membrane association and induction of aberrant protrusions. Autophosphorylation of threonine-440 and threonine-443 in the triple-helical region by the kinase domain blocked the plasma membrane association of TAOK1. These findings define TAOK1 as a plasma membrane remodeling kinase and reveal the underlying mechanisms through which TAOK1 dysfunction may lead to neurodevelopmental disorders.

Phosphoregulation of the septin cytoskeleton in neuronal development and disease.

Septins are highly conserved GTP-binding proteins that oligomerize and form higher order structures. The septin cytoskeleton plays an important role in cellular organization, intracellular transport, and cytokinesis. Kinase-mediated phosphorylation of septins regulates various aspects of their function, localization, and dynamics. Septins are enriched in the mammalian nervous system where they contribute to neurodevelopment and neuronal function. Emerging research has implicated aberrant changes in septin cytoskeleton in several human diseases. The mechanisms through which aberrant phosphorylation by kinases contributes to septin dysfunction in neurological disorders are poorly understood and represent an important question for future research with therapeutic implications. This review summarizes the current state of knowledge of the diversity of kinases that interact with and phosphorylate mammalian septins, delineates how phosphoregulation impacts septin dynamics, and describes how aberrant septin phosphorylation contributes to neurological disorders.

The HSP90 inhibitor 17-AAG induced calcium-mediated apoptosis in filarial parasites.

The available antifilarial medications are effective only against the larval stage of the filarial parasite. As a result, there is a pressing need for an adulticidal drug. The development of drugs requires the identification of molecular targets that are critical for parasite life. In this study, we observed the effect of 17-N-allyl-17-demethoxygeldanamycin on the survival of adult filarial parasites. The 17-N-allyl-17-demethoxygeldanamycin (17-AAG) is a derivative of geldanamycin (GA), which is an inhibitor of heat shock protein (HSP)90. It is less toxic as compared to geldanamycin. The motility and viability of the adult filarial parasite Setaria cervi were decreased on exposure to 17-AAG at 2.5 and 5.0 µM/ml concentrations. The 17-AAG treated parasites showed induction of oxidative stress as evidenced by decreased activity of various antioxidant enzymes like glutathione s-transferase, glutathione reductase, thioredoxin reductase, and an increase in ROS production in comparison to control. Oxidative stress may lead to altered calcium homeostasis. Indeed, in 17-AAG treated worms, there was a rise in calcium in the cytosol and mitochondria, as well as a decrease in the ER. We also observed enhanced activity of phospholipase C in the treated parasite, suggesting the opening of calcium channels located on the ER membrane. ER stress is marked by a reduced level of protein disulfide isomerase. Further, 17-AAG treated worms showed an increase in apoptotic marker enzyme activities like calpain, cyt-c, and caspase-3. The 2D-gel electrophoresis technique showed 142 protein spots in the control and 112 spots in the 17-AAG treated parasite. Thus, 17-AAG induced oxidative stress, and altered calcium, and proteostasis of parasites, which led to apoptosis.

Proteomic Analysis Reveals Differential Protein Expression Induced by Inhibition of Prolyl Oligopeptidase in Filarial Parasites.

Prolyl oligopeptidase (POP) plays a crucial role in the processing and degradation of neuropeptides and regulates inositol trisphosphate (IP3) signaling in mammals. We have reported that POP inhibition leads to IP3-mediated calcium efflux leading to mitochondrial-mediated apoptosis in the filarial parasite Setaria cervi. This study further elucidates the effect of altered calcium homeostasis on the proteome of filarial parasites. Adult parasites were treated with POP's specific inhibitor, Z-Pro-prolinal (ZPP), for 7 h. Cytosolic and mitochondrial proteome was analyzed using 2D gel electrophoresis coupled with MALDI-MS/MS. Phosphoproteins were also analyzed in the cytosolic fraction of the parasites. The phosphoprotein analysis revealed 7, and 9 spots in the cytosolic fraction of control and ZPP-treated parasites, respectively. The two identified protein spots in the treated set were found to be involved in G protein signaling. In cytosolic fraction, 109 and 112 protein spots were observed in control and treated parasites, respectively. Of these, 56 upregulated and 32 downregulated protein spots were observed in the treated set. On the other hand, 50 and 47 protein spots were detected in the mitochondrial fraction of control and treated parasites, respectively. Of these spots, 18 upregulated and 12 down-regulated protein spots were found in treated parasites. In silico analysis showed that the identified proteins were involved in energy metabolism, calcium signaling, stress response, and cytoskeleton organization. These findings correlate with our previous results suggesting the important regulatory role of POP in signaling and different metabolic pathways of filarial parasites.

Antifilarial efficacy of andrographolide: Ex vivo studies on bovine filarial parasite Setaria cervi.

Lymphatic filariasis caused by filarial nematode is an important disease leading to considerable morbidity throughout tropical countries. Even after specific elimination programs, the disease continue to spread in endemic countries. Thus newer therapeutic interventions are urgently needed to control the spread. In the present study, we have seen the effect of andrographolide (andro), a diterpenoid lactone from the leaves of Andrographis paniculata on filarial parasite Setaria cervi. There was time and concentration dependent decrease in motility and viability leading to death of parasite after 6 h of the exposure of andro. Andro showed potential antifilarial activity with an IC50 value of 24.80 µM assessed through MTT assay. There was concentration dependent decrease in the antioxidant enzymes activity and increase in proapoptotic markers after 5 h exposure of andro. Further, molecular docking analysis revealed that andro binds with filarial glutathione-S-transferase at glutathione (GSH) binding site and inhibiting enzyme activity competitively. Andro induced oxidative stress mediated apoptosis in parasites as evidenced by increase in the intracellular reactive oxygen species (ROS) and apoptotic markers.Therefore this study suggested that andro could be further explored as a new antifilarial drug.

Proteomic Identification of Phosphorylation-Dependent Septin 7 Interactors that Drive Dendritic Spine Formation.

Septins are a family of cytoskeletal proteins that regulate several important aspects of neuronal development. Septin 7 (Sept7) is enriched at the base of dendritic spines in excitatory neurons and mediates both spine formation and spine and synapse maturation. Phosphorylation at a conserved C-terminal tail residue of Sept7 mediates its translocation into the dendritic spine head to allow spine and synapse maturation. The mechanistic basis for postsynaptic stability and compartmentalization conferred by phosphorylated Sept7, however, is unclear. We report herein the proteomic identification of Sept7 phosphorylation-dependent neuronal interactors. Using Sept7 C-terminal phosphopeptide pulldown and biochemical assays, we show that the 14-3-3 family of proteins specifically interacts with Sept7 when phosphorylated at the T426 residue. Biochemically, we validate the interaction between Sept7 and 14-3-3 isoform gamma and show that 14-3-3 gamma is also enriched in the mature dendritic spine head. Furthermore, we demonstrate that interaction of phosphorylated Sept7 with 14-3-3 protects it from dephosphorylation, as expression of a 14-3-3 antagonist significantly decreases phosphorylated Sept7 in neurons. This study identifies 14-3-3 proteins as an important physiological regulator of Sept7 function in neuronal development.

TAOK2 is an ER-localized kinase that catalyzes the dynamic tethering of ER to microtubules.

The endoplasmic reticulum (ER) depends on extensive association with the microtubule (MT) cytoskeleton for its structure and mitotic inheritance. However, mechanisms that underlie coupling of ER membranes to MTs are poorly understood. We have identified thousand and one amino acid kinase 2 (TAOK2) as a pleiotropic protein kinase that mediates tethering of ER to MTs. In human cells, TAOK2 localizes in distinct ER subdomains via transmembrane helices and an adjacent amphipathic region. Through its C-terminal tail, TAOK2 directly binds MTs, coupling ER membranes to the MT cytoskeleton. In TAOK2 knockout cells, although ER-membrane dynamics are increased, movement of ER along growing MT plus ends is disrupted. ER-MT tethering is tightly regulated by catalytic activity of TAOK2, perturbation of which leads to defects in ER morphology, association with MTs, and cell division. Our study identifies TAOK2 as an ER-MT tether and reveals a kinase-regulated mechanism for control of ER dynamics.

Kinase Signaling in Dendritic Development and Disease.

Dendrites undergo extensive growth and remodeling during their lifetime. Specification of neurites into dendrites is followed by their arborization, maturation, and functional integration into synaptic networks. Each of these distinct developmental processes is spatially and temporally controlled in an exquisite fashion. Protein kinases through their highly specific substrate phosphorylation regulate dendritic growth and plasticity. Perturbation of kinase function results in aberrant dendritic growth and synaptic function. Not surprisingly, kinase dysfunction is strongly associated with neurodevelopmental and psychiatric disorders. Herein, we review, (a) key kinase pathways that regulate dendrite structure, function and plasticity, (b) how aberrant kinase signaling contributes to dendritic dysfunction in neurological disorders and (c) emergent technologies that can be applied to dissect the role of protein kinases in dendritic structure and function.

Glial ensheathment of the somatodendritic compartment regulates sensory neuron structure and activity.

Sensory neurons perceive environmental cues and are important of organismal survival. Peripheral sensory neurons interact intimately with glial cells. While the function of axonal ensheathment by glia is well studied, less is known about the functional significance of glial interaction with the somatodendritic compartment of neurons. Herein, we show that three distinct glia cell types differentially wrap around the axonal and somatodendritic surface of the polymodal dendritic arborization (da) neuron of the Drosophila peripheral nervous system for detection of thermal, mechanical, and light stimuli. We find that glial cell-specific loss of the chromatin modifier gene dATRX in the subperineurial glial layer leads to selective elimination of somatodendritic glial ensheathment, thus allowing us to investigate the function of such ensheathment. We find that somatodendritic glial ensheathment regulates the morphology of the dendritic arbor, as well as the activity of the sensory neuron, in response to sensory stimuli. Additionally, glial ensheathment of the neuronal soma influences dendritic regeneration after injury.

Meeting report - shining light on septins.

Septins are enigmatic proteins; they bind GTP and assemble together like molecular Lego blocks to form intracellular structures of varied shapes such as filaments, rings and gauzes. To shine light on the biological mysteries of septin proteins, leading experts in the field came together for the European Molecular Biology Organization (EMBO) workshop held from 8-11 October 2017 in Berlin. Organized by Helge Ewers (Freie Universität, Berlin, Germany) and Serge Mostowy (Imperial College, London, UK), the workshop convened at the Harnack-Haus, a historic hub of scientific discourse run by the Max Planck Society.

Cellular Phenotypes in Human iPSC-Derived Neurons from a Genetic Model of Autism Spectrum Disorder.

A deletion or duplication in the 16p11.2 region is associated with neurodevelopmental disorders, including autism spectrum disorder and schizophrenia. In addition to clinical characteristics, carriers of the 16p11.2 copy-number variant (CNV) manifest opposing neuroanatomical phenotypes-e.g., macrocephaly in deletion carriers (16pdel) and microcephaly in duplication carriers (16pdup). Using fibroblasts obtained from 16pdel and 16pdup carriers, we generated induced pluripotent stem cells (iPSCs) and differentiated them into neurons to identify causal cellular mechanisms underlying neurobiological phenotypes. Our study revealed increased soma size and dendrite length in 16pdel neurons and reduced neuronal size and dendrite length in 16pdup neurons. The functional properties of iPSC-derived neurons corroborated aspects of these contrasting morphological differences that may underlie brain size. Interestingly, both 16pdel and 16pdup neurons displayed reduced synaptic density, suggesting that distinct mechanisms may underlie brain size and neuronal connectivity at this locus.

TAOK2 Kinase Mediates PSD95 Stability and Dendritic Spine Maturation through Septin7 Phosphorylation.

Abnormalities in dendritic spines are manifestations of several neurodevelopmental and psychiatric diseases. TAOK2 is one of the genes in the 16p11.2 locus, copy number variations of which are associated with autism and schizophrenia. Here, we show that the kinase activity of the serine/threonine kinase encoded by TAOK2 is required for spine maturation. TAOK2 depletion results in unstable dendritic protrusions, mislocalized shaft-synapses, and loss of compartmentalization of NMDA receptor-mediated calcium influx. Using chemical-genetics and mass spectrometry, we identified several TAOK2 phosphorylation targets. We show that TAOK2 directly phosphorylates the cytoskeletal GTPase Septin7, at an evolutionary conserved residue. This phosphorylation induces translocation of Septin7 to the spine, where it associates with and stabilizes the scaffolding protein PSD95, promoting dendritic spine maturation. This study provides a mechanistic basis for postsynaptic stability and compartmentalization via TAOK2-Sept7 signaling, with implications toward understanding the potential role of TAOK2 in neurological deficits associated with the 16p11.2 region.

Epidermis-Derived Semaphorin Promotes Dendrite Self-Avoidance by Regulating Dendrite-Substrate Adhesion in Drosophila Sensory Neurons.

Precise patterning of dendritic arbors is critical for the wiring and function of neural circuits. Dendrite-extracellular matrix (ECM) adhesion ensures that the dendrites of Drosophila dendritic arborization (da) sensory neurons are properly restricted in a 2D space, and thereby facilitates contact-mediated dendritic self-avoidance and tiling. However, the mechanisms regulating dendrite-ECM adhesion in vivo are poorly understood. Here, we show that mutations in the semaphorin ligand sema-2b lead to a dramatic increase in self-crossing of dendrites due to defects in dendrite-ECM adhesion, resulting in a failure to confine dendrites to a 2D plane. Furthermore, we find that Sema-2b is secreted from the epidermis and signals through the Plexin B receptor in neighboring neurons. Importantly, we find that Sema-2b/PlexB genetically and physically interacts with TORC2 complex, Tricornered (Trc) kinase, and integrins. These results reveal a novel role for semaphorins in dendrite patterning and illustrate how epidermal-derived cues regulate neural circuit assembly.

RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization.

Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth.

Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis.

MST3 kinase phosphorylates TAO1/2 to enable Myosin Va function in promoting spine synapse development.

Mammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS.

Golgin160 recruits the dynein motor to position the Golgi apparatus.

Membrane motility is a fundamental characteristic of all eukaryotic cells. One of the best-known examples is that of the mammalian Golgi apparatus, where constant inward movement of Golgi membranes results in its characteristic position near the centrosome. While it is clear that the minus-end-directed motor dynein is required for this process, the mechanism and regulation of dynein recruitment to Golgi membranes remains unknown. Here, we show that the Golgi protein golgin160 recruits dynein to Golgi membranes. This recruitment confers centripetal motility to membranes and is regulated by the GTPase Arf1. Further, during cell division, motor association with membranes is regulated by the dissociation of the receptor-motor complex from membranes. These results identify a cell-cycle-regulated membrane receptor for a molecular motor and suggest a mechanistic basis for achieving the dramatic changes in organelle positioning seen during cell division.

Golgi positioning.

The Golgi apparatus in mammalian cells is positioned near the centrosome-based microtubule-organizing center (Fig. 1). Secretory cargo moves inward in membrane carriers for delivery to Golgi membranes in which it is processed and packaged for transport outward to the plasma membrane. Cytoplasmic dynein motor proteins (herein termed dynein) primarily mediate inward cargo carrier movement and Golgi positioning. These motors move along microtubules toward microtubule minus-ends embedded in centrosomes. Centripetal motility is controlled by a host of regulators whose precise functions remain to be determined. Significantly, a specific Golgi receptor for dynein has not been identified. This has impaired progress toward elucidation of membrane-motor-microtubule attachment in the periphery and, after inward movement, recycling of the motor for another round. Pericentrosomal positioning of the Golgi apparatus is dynamic. It is regulated during critical cellular processes such as mitosis, differentiation, cell polarization, and cell migration. Positioning is also important as it aligns the Golgi along an axis of cell polarity. In certain cell types, this promotes secretion directed to the proximal plasma membrane domain thereby maintaining specializations critical for diverse processes including wound healing, immunological synapse formation, and axon determination.