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It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope., Author summary During all herpesvirus infections, the nuclear envelope undergoes deformation in order to enable viral capsids assembled within the nucleus of the infected cell to gain access to the cytoplasm for further maturation and spread to neighbouring cells. These nuclear envelope deformations are orchestrated by the viral nuclear egress complex (NEC), which, in HSV, is composed of two viral proteins, pUL31 and pUL34. How the membrane-deformation activity of the NEC is controlled during infection is incompletely understood. The studies in this communication reveal that the phosphorylation status of pUL31 and pUL34 can determine the nature of nuclear envelope deformations and that the viral protein pUL21 can modulate the phosphorylation status of both NEC components. These findings provide an explanation for why HSV-2 strains lacking pUL21 are defective in nuclear egress. A thorough understanding of how NEC activity is controlled during infection may yield strategies to disrupt this fundamental step in the herpesvirus lifecycle, providing the basis for novel antiviral strategies.

When cells with excess DNA, such as tetraploid cells, undergo cell division, it can contribute to cellular transformation via asymmetrical chromosome segregation-generated genetic diversity. Cell cycle progression of tetraploid cells is suppressed by large tumor suppressor 2 (LATS2) kinase-induced inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP). We recently reported that the oncogene v-Src induces tetraploidy and promotes cell cycle progression of tetraploid cells by suppressing LATS2 activity. We explore here the mechanism by which v-Src suppresses LATS2 activity and the role of LATS2 in v-Src-expressing cells. LATS2 was directly phosphorylated by v-Src and the proto-oncogene c-Src, resulting in decreased LATS2 kinase activity. This kinase-deficient LATS2 accumulated in a YAP transcriptional activity-dependent manner, and knockdown of either LATS2 or the LATS2-binding partner moesin-ezrin-radixin-like protein (Merlin) accelerated v-Src-induced membrane bleb formation. Upon v-Src expression, the interaction of Merlin with LATS2 was increased possibly due to a decrease in Merlin phosphorylation at Ser518, the dephosphorylation of which is required for the open conformation of Merlin and interaction with LATS2. LATS2 was colocalized with Merlin at the plasma membrane in a manner that depends on the Merlin-binding region of LATS2. The bleb formation in v-Src-expressing and LATS2-knockdown cells was rescued by the reexpression of wild-type or kinase-dead LATS2 but not the LATS2 mutant lacking the Merlin-binding region. These results suggest that the kinase-deficient LATS2 plays a role with Merlin at the plasma membrane in the maintenance of cortical rigidity in v-Src-expressing cells, which may cause tumor suppression.

The evolutionarily highly conserved SNF1-related protein kinase (SnRK1) protein kinase is a metabolic master regulator in plants, balancing the critical energy consumption between growth- and stress response-related metabolic pathways. While the regulation of the mammalian [AMP-activated protein kinase (AMPK)] and yeast (SNF1) orthologues of SnRK1 is well-characterised, the regulation of SnRK1 kinase activity in plants is still an open question. Here we report that the activity and T-loop phosphorylation of AKIN10, the kinase subunit of the SnRK1 complex, is regulated by the redox status. Although this regulation is dependent on a conserved cysteine residue, the underlying mechanism is different to the redox regulation of animal AMPK and has functional implications for the regulation of the kinase complex in plants under stress conditions.

Deoxyguanosine kinase (DGUOK) deficiency causes mtDNA depletion and mitochondrial dysfunction. We reported long survival of DGUOK knockout (Dguok−/−) mice despite low (

Corneal endothelial cell (CEC) dysfunction causes corneal edema and severe visual impairment that require transplantation to restore vision. To address the unmet need of organ shortage, descemetorhexis without endothelial keratoplasty has been specifically employed to treat early stage Fuchs endothelial corneal dystrophy, which is pathophysiologically related to oxidative stress and exhibits centrally located corneal guttae. After stripping off central Descemet's membrane, rho-associated protein kinase (ROCK) inhibitor has been found to facilitate CEC migration, an energy-demanding task, thereby achieving wound closure. However, the correlation between ROCK inhibition and the change in bioenergetic status of CECs remained to be elucidated. Through transcriptomic profiling, we found that the inhibition of ROCK activity by the selective inhibitor, ripasudil or Y27632, promoted enrichment of oxidative phosphorylation (OXPHOS) gene set in bovine CECs (BCECs). Functional analysis revealed that ripasudil, a clinically approved anti-glaucoma agent, enhanced mitochondrial respiration, increased spare respiratory capacity, and induced overexpression of electron transport chain components through upregulation of AMP-activated protein kinase (AMPK) pathway. Accelerated BCEC migration and in vitro wound healing by ripasudil were diminished by OXPHOS and AMPK inhibition, but not by glycolysis inhibition. Correspondingly, lamellipodial protrusion and actin assembly that were augmented by ripasudil became reduced with additional OXPHOS or AMPK inhibition. These results indicate that ROCK inhibition induces metabolic reprogramming toward OXPHOS to support migration of CECs.

Brain derived neurotrophic factor (BDNF) acting through the tropomyosin-related kinase receptor B (TrkB) enhances neuromuscular transmission in the diaphragm muscle of adult mice, reflecting presynaptic effects. With aging, BDNF enhancement of neuromuscular transmission is lost. We hypothesize that disrupting BDNF/TrkB signaling in early old age will reveal a period of susceptibility evident by morphological changes at neuromuscular junctions (NMJ). Adult, male TrkB(F616A) mice (n=25) at 6 and 18 months of age, were used to examine the structural properties of diaphragm muscle NMJs (n=1097). Confocal microscopy was used to compare pre- and post-synaptic morphology and denervation following a 7 day treatment with the phosphoprotein phosphatase-1 derivative 1NMPP1, which inhibits TrkB kinase activity in TrkB(F616A) mice vs. vehicle treatment. In early old age (18 months), presynaptic terminal volume decreased compared to 6 month old diaphragm NMJs (~20%). Inhibition of TrkB kinase activity significantly decreased the presynaptic terminal volume (~20%) and motor end-plate 2D planar area (~10%), independent of age group. Inhibition of TrkB kinase activity in early old age significantly reduced overlap of pre- and post-synaptic structures and increased the proportion of denervated NMJs (to ~20%). Collectively these results support a period of susceptibility in early old age when BDNF/TrkB signaling at diaphragm NMJs supports the maintenance of NMJs structure and muscle innervation.

Mammalian Toll-like receptors (TLR) recognize microbial products and elicit transient immune responses that protect the infected host from disease. TLR4--which signals from both plasma and endosomal membranes--is activated by bacterial lipopolysaccharides (LPS) and induces many cytokine genes, the prolonged expression of which causes septic shock in mice. We report here that the expression of some TLR4-induced genes in myeloid cells requires the protein kinase activity of the epidermal growth factor receptor (EGFR). EGFR inhibition affects TLR4-induced responses differently depending on the target gene. The induction of interferon-β (IFN-β) and IFN-inducible genes is strongly inhibited, whereas TNF-α induction is enhanced. Inhibition is specific to the IFN-regulatory factor (IRF)-driven genes because EGFR is required for IRF activation downstream of TLR--as is IRF co-activator β-catenin--through the PI3 kinase/AKT pathway. Administration of an EGFR inhibitor to mice protects them from LPS-induced septic shock and death by selectively blocking the IFN branch of TLR4 signaling. These results demonstrate a selective regulation of TLR4 signaling by EGFR and highlight the potential use of EGFR inhibitors to treat septic shock syndrome.

Mutations in the LRRK2 gene cause autosomal dominant Parkinson's disease. LRRK2 encodes a multi-domain protein containing a Ras-of-complex (Roc) GTPase domain, a C-terminal of Roc domain and a protein kinase domain. LRRK2 can function as a GTPase and protein kinase, although the interplay between these two enzymatic domains is poorly understood. Although guanine nucleotide binding is critically required for the kinase activity of LRRK2, the contribution of GTP hydrolysis is not known. In general, the molecular determinants regulating GTPase activity and how the GTPase domain contributes to the properties of LRRK2 remain to be clarified. Here, we identify a number of synthetic missense mutations in the GTPase domain that functionally modulate GTP binding and GTP hydrolysis and we employ these mutants to comprehensively explore the contribution of GTPase activity to the kinase activity and cellular phenotypes of LRRK2. Our data demonstrate that guanine nucleotide binding and, to a lesser extent, GTP hydrolysis are required for maintaining normal kinase activity and both activities contribute to the GTP-dependent activation of LRRK2 kinase activity. Guanine nucleotide binding but not GTP hydrolysis regulates the dimerization, structure and stability of LRRK2. Furthermore, GTP hydrolysis regulates the LRRK2-dependent inhibition of neurite outgrowth in primary cortical neurons but is unable to robustly modulate the effects of the familial G2019S mutation. Our study elucidates the role of GTPase activity in regulating kinase activity and cellular phenotypes of LRRK2 and has important implications for the validation of the GTPase domain as a molecular target for attenuating LRRK2-mediated neurodegeneration.

JAK2 is cytokine-activated non-receptor tyrosine kinase. Although JAK2 is mainly localized at the plasma membrane, it is also present on the centrosome. In this study, we demonstrated that JAK2 localization to the centrosome depends on the SH2 domain and intact kinase activity. We created JAK2 mutants deficient in centrosomal localization ΔSH2, K882E and (ΔSH2, K882E). We showed that JAK2 WT clone strongly enhances cell proliferation as compared to control cells while JAK2 clones ΔSH2, K882E and (ΔSH2, K882E) proliferate slower than JAK2 WT cells. These mutant clones also progress much slower through the cell cycle as compared to JAK2 WT clone and the enhanced proliferation of JAK2 WT cells is accompanied by increased S −> G2 progression. Both the SH2 domain and the kinase activity of JAK2 play a role in prolactin-dependent activation of JAK2 substrate STAT5. We showed that JAK2 is an important regulator of centrosome function as the SH2 domain of JAK2 regulates centrosome amplification. The cells overexpressing ΔSH2 and (ΔSH2, K-E) JAK2 have almost three-fold the amplified centrosomes of WT cells. In contrast, the kinase activity of JAK2 is dispensable for centrosome amplification. Our observations provide novel insight into the role of SH2 domain and kinase activity of JAK2 in centrosome localization of JAK2 and in the regulation of cell growth and centrosome biogenesis.

Made available in DSpace on 2020-12-12T00:57:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) There is a gap in the knowledge regarding the regulation of glucose uptake in skeletal muscle during the development of insulin resistance in the elderly. Rho-Kinase (Rock) signaling has been demonstrated as a crucial mechanism related to glucose metabolism and insulin sensitivity in skeletal muscle. This kinase is involved in the insulin receptor substrate 1 (IRS1) phosphorylation, leading to glucose uptake stimulation in the skeletal muscle; however, the mechanisms elucidating the role of Rock regulation in the context of advanced ages are still limited. In this study, we submitted old Fischer 344 rats to short-term treadmill physical exercise protocol (5 days) and evaluated the glucose tolerance and proteins involved with Rock/insulin signaling in the skeletal muscle. Compared to young rats, the old rats showed glucose intolerance, hyperinsulinemia, and decreased phosphorylation in the proteins related to the insulin signaling pathway in the skeletal muscle, without changes in body mass and adiposity. Otherwise, when these rats were submitted to physical exercise, it was found decreased fasting glucose, higher glucose tolerance, decreased insulinemia, and upregulation of Rock2/pIRS1/pAkt/pGSK3β/GLUT4 pathway in the skeletal muscle. In summary, the aging process did not change Rock signaling, but the physical exercise was able to increase Rock2 content and insulin signaling pathway in the skeletal muscle. This finding suggests the benefic role of physical exercise to advanced ages, promoting insulin-sensitive effects with Rho-kinase contribution. Laboratory of Molecular Biology of Exercise University of Campinas (UNICAMP) Center for Neuroscience and Cell Biology (CNC.IBILI) University of Coimbra Postgraduate Program in Rehabilitation and Functional Performance Ribeirão Preto Medical School USP School of Physical Education and Sport of Ribeirão Preto University of São Paulo (USP) OCRC - Obesity and Comorbidities Research Center University of Campinas (UNICAMP) Postgraduate Program in Motor Science - São Paulo State University (UNESP) CEPECE - Center of Research in Sport Sciences School of Applied Sciences University of Campinas (UNICAMP) Exercise Cell Biology Lab University of Campinas (UNICAMP) Laboratory of Nutritional Genomics University of Campinas (UNICAMP) Postgraduate Program in Motor Science - São Paulo State University (UNESP) CAPES: 001 FAPESP: 2015/26000-2 FAPESP: 2016/18488-8 FAPESP: 2018/20872-6 CNPq: 306535/2017-3 CNPq: 442542/2014-3

Testis specific serine/threonine protein kinase 4 (TSSK4) belongs to the TSSK family, and its members play an important role in spermatogenesis and/or spermiogenesis. Mouse TSSK4 has been reported to be expressed exclusively in the testis and can maintain its kinase activity through autophosphorylation at Thr-197. However, its biological function remains poorly understood. Here we found that GFP-TSSK4-overexpressed HeLa cells showed apoptotic bodies, indicating TSSK4 can lead to apoptosis in vitro. Furthermore, TSSK4 induced apoptosis in different cell lines including HeLa, Cos-7 and H1299 tested by flow cytometry but not its kinase-dead mutant TSSK4-K54M. TSSK4 knockout mice showed increased testes weight and decreased apoptotic spermatogonia and spermatocytes at 21st day after birth tested by TUNEL technology. So TSSK4 was able to induce cell apoptosis in vitro depending on its kinase activity, which leads to abnormal testes weight and apoptosis, shedding light on its function in the process of spermatogenesis and/or spermiogenesis.

The voltage-gated potassium channel Kv3.4 is a crucial regulator of nociceptive signaling in the dorsal root ganglion (DRG) and the dorsal horn of the spinal cord. Moreover, Kv3.4 dysfunction has been linked to neuropathic pain. Although kinases and phosphatases can directly modulate Kv3.4 gating, the signaling mechanisms regulating the expression and stability of the Kv3.4 protein are generally unknown. We explored a potential role of PKCe and found an unexpected interaction that has a positive effect on Kv3.4 expression. Co-immunoprecipitation studies revealed a physical association between PKCe and Kv3.4 in both heterologous cells and rat DRG neurons. Furthermore, in contrast to the wild-type and constitutively active forms of PKCe, expression of a catalytically inactive form of the enzyme inhibits Kv3.4 expression and membrane localization through a dominant negative effect. Co-expression of Kv3.4 with the wild-type, constitutively active, or catalytically inactive forms of PKCe had no significant effects on Kv3.4 gating. These results suggest that a novel physical interaction of the Kv3.4 channel with functional PKCe primarily determines its stability and localization in DRG neurons. This interaction is akin to those of previously identified accessory ion channel proteins, which could be significant in neural tissues where Kv3.4 regulates electrical signaling.

Amiloride is widely used in clinical practice as a diuretic and is known to interact with the epithelial sodium channel and acid‑sensing ion channel proteins, as well as Na+/H+ antiporters and Na+/Ca2+ exchangers. The aim of the present study was to examine the effects of amiloride on receptor activator of nuclear factor‑κB ligand (RANKL)‑induced osteoclastogenesis and to elucidate the underlying mechanisms in the RAW264.7 murine macrophage cell line. The number of tartrate‑resistant acid phosphatase (TRAP)‑positive multinucleated cells were counted and the bone resorption area was estimated. In addition the expression levels of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) mRNA and osteoclast‑specific genes, including TRAP, matrix metalloproteinase 9, cathepsin K and osteoclast‑associated receptor, were examined using reverse transcription‑quantitative polymerase chain reaction. The nuclear factor‑κB (NF‑κB) and mitogen‑activated protein kinase (MAPK) signaling pathways were also investigated using western blotting. The results showed that amiloride significantly reduced the number of TRAP‑positive multinucleated cells as well as the bone resorption area. Amiloride also downregulated the expression of NFATc1 mRNA and inhibited the expression of osteoclast‑specific genes. A possible underlying mechanism may be that amiloride suppresses the degradation of the inhibitor of NF‑κB and blocks the activation of c‑Jun N‑terminal kinase, extracellular signal‑regulated kinase and p38, thus implicating the NF‑κB and MAPK pathway is this process. In conclusion, the current data suggest that amiloride is a strong inhibitor of osteoclast differentiation, indicating a novel indication for amiloride in the treatment of bone‑loss‑related diseases.

NME1 (nonmetastatic expressed 1) gene, which encodes nucleoside diphosphate kinase (NDPK) A [also known as nonmetastatic clone 23 (NM23)-H1 in humans and NM23-M1 in mice], is a suppressor of metastasis, but several lines of evidence-mostly from plants-also implicate it in the regulation of the oxidative stress response. Here, our aim was to investigate the physiologic relevance of NDPK A with respect to the oxidative stress response in mammals and to study its molecular basis. NME1-knockout mice died sooner, suffered greater hepatocyte injury, and had lower superoxide dismutase activity than did wild-type (WT) mice in response to paraquat-induced acute oxidative stress. Deletion of NME1 reduced total NDPK activity and exacerbated activation of the stress-related MAPK, JNK, in the liver in response to paraquat. In a mouse transformed hepatocyte cell line and in primary cultures of normal human keratinocytes, MAPK activation in response to H2O2 and UVB, respectively, was dampened by expression of NM23-M1/NM23-H1, dependent on its NDPK catalytic activity. Furthermore, excess or depletion of NM23-M1/NM23-H1 NDPK activity did not affect the intracellular bulk concentration of nucleoside di- and triphosphates. NME1-deficient mouse embryo fibroblasts grew poorly in culture, were more sensitive to stress than WT fibroblasts, and did not immortalize, which suggested that they senesce earlier than do WT fibroblasts. Collectively, these results indicate that the NDPK activity of NM23-M1/NM23-H1 protects cells from acute oxidative stress by inhibiting activation of JNK in mammal models.-Peuchant, E., Bats, M.-L., Moranvillier, I., Lepoivre, M., Guitton, J., Wendum, D., Lacombe, M.-L., Moreau-Gaudry, F., Boissan, M., Dabernat, S. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons., Author summary Staphylococcus aureus is a major human pathogen, and has become a significant worldwide health concern due to the rapid emergence of antibiotic resistant strains. Like most bacteria, S. aureus adapts to its environment by adjusting its genetic expression through sensing and regulatory systems. We show here that the SpdC membrane protein is a novel virulence factor of S. aureus, controlling biofilm formation and pathogenesis. We show that SpdC interacts with the WalK histidine kinase to inhibit activity of the WalKR two-component system. SpdC also interacts with nine other histidine kinases of S. aureus, suggesting it acts as a pleiotropic global regulator.

Nucleoside diphosphate kinase (NDK), which has the same sequence as oncoprotein (OP) in humans, can induce nucleoside triphosphates in DNA replication by maintenance of the deoxynucleotide triphosphate (dNTP’s) and is known to be regulated by viral infection in the shrimp Litopenaeus vannamei. This paper describes the relationship between NDK and white spot syndrome virus (WSSV) infection. The recombinant NDK was produced by a prokaryotic expression system. WSSV copy numbers and mRNA levels of IE1 and VP28 were significantly increased in shrimp injected with recombinant NDK at 72 h after WSSV infection. After synthesizing dsRNA-NDK and confirming the efficacy of NDK silencing, we recorded the cumulative mortality of WSSV-infected shrimp injected with NDK and dsRNA-NDK. A comparison between the results demonstrated that silencing NDK delayed the death of shrimps. These findings indicate that NDK has an important role influencing the replication of WSSV replication in shrimp. Furthermore, NDK may have potential target as a new therapeutic strategy against WSSV infection in shrimp.

HMGB2, a DNA-binding protein, highly expresses during embryogenesis and plays an important role in development of some organs and tissues. However, it remains to be further investigated weather HMGB2 influences muscle development. In this work, we identified HMGB2 as an essential factor in myogenesis. Compared to wild type (WT) mice, body weights of systemic hmgb2 homozygous knockout (hmgb2-/- ) mice especially males were reduced. Diameter and cross-section area of tibialis anterior (TA) muscle fibers as well as expression of Myogenin and MyHC were all decreased in hmgb2-/- mice. CTX injury model revealed that HMGB2 was required for satellite cell proliferation and muscle regeneration. Moreover, HMGB2 interacted with S6K1 and regulated the kinase activity of S6K1 during cell proliferation. Knockdown and inactivation of S6K1 in C2C12 cells both resulted in impaired proliferation and differentiation. Furthermore, expression of cyclin D1 and Myf5 were both decreased when HMGB2 or S6K1 were knocked down and kinase activity of S6K1 was inhibited. These results indicate that HMGB2 is required for skeletal muscle development and regeneration, and HMGB2 maintains proliferation of myoblasts through regulating kinase activity of S6K1.

Activated and promoter-bound heat-shock transcription factor 1 (HSF1) induces RNA polymerase II recruitment upon heat shock, and this is facilitated by the core Mediator in Drosophila and yeast. Another Mediator module, CDK8 kinase module (CKM), consisting of four subunits including MED12 and CDK8, plays a negative or positive role in the regulation of transcription; however, its involvement in HSF1-mediated transcription remains unclear. We herein demonstrated that HSF1 interacted with MED12 and recruited MED12 and CDK8 to the HSP70 promoter during heat shock in mammalian cells. The kinase activity of CDK8 (and its paralog CDK19) promoted HSP70 expression partly by phosphorylating HSF1-S326 and maintained proteostasis capacity. These results indicate an important role for CKM in the protection of cells against proteotoxic stress.

Cognitive deficit is a prevalent and underestimated complication of diabetes, and the underlying cellular and molecular mechanisms are not well understood. Aberrant activity of cyclin-dependent kinase (Cdk)5 is implicated in a number of neurodegenerative diseases. The present study examined the role of Cdk5 in the progression of diabetes-related cognitive deficits. We showed that the Cdk5 protein expression and kinase activity were significantly increased in diabetic mice at 16 wk. In primary cultured hippocampal neurons exposed to 30 mM glucose, Cdk5 protein and kinase activity were also elevated in a time-dependent manner. Moreover, the high glucose exposure led to an aberrant Cdk5 activation due to its activator p25 that was cleaved from p35 by calpain. Both in diabetic mice and in cultured hippocampal neurons exposed to high glucose, inhibition of Cdk5 activity with roscovitine (Ros) or short hairpin RNA (shRNA) decreased the protein levels of cleaved caspase-3 and the ratio of Bax and Bcl-2. The apoptotic rate detected by TUNEL in vivo or Annexin V and propidium iodide staining for flow cytometry in vitro also had obvious reduction. In addition, high glucose exposure resulted in the increase of phosphorylated (phospho)-MAPK kinase (MKK)6, phospho-p38, and c-Jun, which were rescued by Ros or Cdk5 shRNA. It is more important that the cognitive deficits of diabetic mice were also effectively alleviated by Ros. These results indicate that aberrant Cdk5 activity triggered hippocampal neuron apoptosis by activating MKK6/p38 MAPK cascade in hyperglycemia. Inhibition of Cdk5 overactivation attenuates neuronal apoptosis and cognitive deficits and contributes to the relief of diabetic neurotoxicity in the brain.-Liu, W., Zhou, Y., Liang, R., Zhang, Y. Inhibition of cyclin-dependent kinase 5 activity alleviates diabetes-related cognitive deficits.

The widely conserved kinase Aurora B regulates important events during cell division. Surprisingly, recent work has uncovered a few functions of Aurora-family kinases that do not require kinase activity. Thus, understanding this important class of cell cycle regulators will require strategies to distinguish kinase-dependent from independent functions. Here, we address this need in C. elegans by combining germline-specific, auxin-induced Aurora B (AIR-2) degradation with the transgenic expression of kinase-inactive AIR-2. Through this approach, we find that kinase activity is essential for AIR-2’s major meiotic functions and also for mitotic chromosome segregation. Moreover, our analysis revealed insight into the assembly of the ring complex (RC), a structure that is essential for chromosome congression in C. elegans oocytes. AIR-2 localizes to chromosomes and recruits other components to form the RC. However, we found that while kinase-dead AIR-2 could load onto chromosomes, other components were not recruited. This failure in RC assembly appeared to be due to a loss of RC SUMOylation, suggesting that there is crosstalk between SUMOylation and phosphorylation in building the RC and implicating AIR-2 in regulating the SUMO pathway in oocytes. Similar conditional depletion approaches may reveal new insights into other cell cycle regulators., Author summary During cell division, chromosomes must be accurately partitioned to ensure the proper distribution of genetic material. In mitosis, chromosomes are duplicated once and then divided once, generating daughter cells with the same amount of genetic material as the original cell. Conversely, during meiosis chromosomes are duplicated once and divided twice, to cut the chromosome number in half to generate eggs and sperm. One important protein that is required for both mitotic and meiotic chromosome segregation is the kinase Aurora B, which phosphorylates a variety of other cell division proteins. However, previous research has shown that some kinases have functions that are independent of their ability to phosphorylate other proteins. Thus, fully understanding how Aurora B regulates cell division requires methods to test whether its various functions require kinase activity. We designed and implemented such a strategy in the model organism C. elegans, by depleting Aurora B from meiotically and mitotically-dividing cells, leaving in place a kinase-inactive version. This work has lent insight into how Aurora B regulates cell division in C. elegans, and also serves as a proof of principle for our approach, which can now be applied to study other essential cell division kinases.

The PKA-inhibitor (PKI) family members PKIα, PKIβ, and PKIγ bind with high affinity to PKA and block its kinase activity, modulating the extent, and duration of PKA-mediated signaling events. While PKA is a well-known regulator of physiological and oncogenic events, the role of PKI proteins in these pathways has remained elusive. Here, by measuring activation of the MAPK pathway downstream of GPCR-Gαs-cAMP signaling, we show that the expression levels of PKI proteins can alter the balance of activation of two major cAMP targets: PKA and EPAC. Our results indicate that PKA maintains repressive control over MAPK signaling as well as a negative feedback on cAMP concentration. Overexpression of PKI and its subsequent repression of PKA dysregulates these signaling pathways, resulting in increased intracellular cAMP, and enhanced activation of EPAC and MAPK. We also find that amplifications of PKIA are common in prostate cancer and are associated with reduced progression free survival. Depletion of PKIA in prostate cancer cells leads to reduced migration, increased sensitivity to anoikis and reduced tumor growth. By altering PKA activity PKI can act as a molecular switch, driving GPCR-Gαs-cAMP signaling toward activation of EPAC-RAP1 and MAPK, ultimately modulating tumor growth.

The study aimed to identify differentially expressed microRNAs (miRNAs/miRs) and explore the mechanisms governing impaired memory and learning ability in developing brains exposed to sevoflurane. A total of six 7‑day‑old male ICR mice were randomly assigned into the sevoflurane anesthesia group (treated with 2.4% sevoflurane) or control group (treated with normal saline solution at the same dose). After 14 days, the mice were subjected to a Morris water maze experiment. Then, the animals were sacrificed and hippocampus tissues were isolated. RNAs in hippocampus tissues were sequenced and the differential miRNA expression profiles were identified by a bioinformatics approach. The learning and memory function of mice were significantly affected by sevoflurane exposure. A total of 18 miRNAs were found to be significantly affected by sevoflurane administration. Their target genes clustered into different functional groups, such as 'dephosphorylation', 'vesicle localization' and the 'Wnt signaling pathway'. miR‑101b‑3p was closely related with 'chromatin binding' and 'protein serine/threonine kinase activity'. The most represented pathways for miRNAs included 'neuroactive ligand‑receptor interaction' (miR‑1187), 'long‑term depression' (miR‑425‑5p), 'FoxO signaling pathway' (miR‑425‑5p) and the 'neurotrophin signaling pathway' (miR‑467a‑3p). miR‑467a‑3p (degree=89), miR‑101b‑3p (degree=59), and miR‑1187 (degree=51) were the hub nodes in the miRNA regulatory network. The Wnt signaling pathway, miR‑467a‑3p, miR‑1187 and miR‑101b‑3p may be therapeutic targets for preventing cognitive impairments induced by sevoflurane.

In tumor necrosis factor (TNF) signaling, phosphorylation and activation of receptor interacting protein kinase 1 (RIPK1) by upstream kinases is an essential checkpoint in the suppression of TNF-induced cell death. Thus, discovery of pharmacological agents targeting RIPK1 may provide new strategies for improving the therapeutic efficacy of TNF. In this study, we found that 3-O-acetylrubianol C (3AR-C), an arborinane triterpenoid isolated from Rubia philippinesis, promoted TNF-induced apoptotic and necroptotic cell death. To identify the molecular mechanism, we found that in mouse embryonic fibroblasts, 3AR-C drastically upregulated RIPK1 kinase activity by selectively inhibiting IKKβ. Notably, 3AR-C did not interfere with IKKα or affect the formation of the TNF receptor1 (TNFR1) complex-I. Moreover, in human cancer cells, 3AR-C was only sufficient to sensitize TNF-induced cell death when c-FLIPL expression was downregulated to facilitate the formation of TNFR1 complex-II and necrosome. Taken together, our study identified a novel arborinane triterpenoid 3AR-C as a potent activator of TNF-induced cell death via inhibition of IKKβ phosphorylation and promotion of the cytotoxic potential of RIPK1, thus providing a rationale for further development of 3AR-C as a selective IKKβ inhibitor to overcome TNF resistance in cancer therpay.

Ginsenoside Re (G-Re) is a panaxatriol saponin and one of the pharmacologically active natural constituents of ginseng (Panax ginseng C.A. Meyer). G-Re has antioxidant, anti-inflammatory and antidiabetic effects. The present study aimed to investigate the effects of G-Re on neuroinflammatory responses in lipopolysaccharide (LPS)-stimulated microglia and its protective effects on hippocampal neurons. Cytokine levels were measured using ELISA and reactive oxygen species (ROS) levels were assessed using flow cytometry and fluorescence microscopy. Protein levels of inflammatory molecules and kinase activity were assessed by western blotting. Cell viability was assessed by MTT assay; apoptosis was estimated by Annexin V apoptosis assay. The results revealed that G-Re significantly inhibited the production of IL-6, TNF-α, nitric oxide (NO) and ROS in BV2 microglial cells, and that of NO in mouse primary microglia, without affecting cell viability. G-Re also inhibited the nuclear translocation of NF-κB, and phosphorylation and degradation of IκB-α. In addition, G-Re dose-dependently suppressed LPS-mediated phosphorylation of Ca2+/calmodulin-dependent protein kinase (CAMK)2, CAMK4, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinases (JNK). Moreover, the conditioned medium from LPS-stimulated microglial cells induced HT22 hippocampal neuronal cell death, whereas that from microglial cells incubated with both LPS and G-Re ameliorated HT22 cell death in a dose-dependent manner. These results suggested that G-Re suppressed the production of pro-inflammatory mediators by blocking CAMK/ERK/JNK/NF-κB signaling in microglial cells and protected hippocampal cells by reducing these inflammatory and neurotoxic factors released from microglial cells. The present findings indicated that G-Re may be a potential treatment option for neuroinflammatory disorders and could have therapeutic potential for various neurodegenerative diseases.

CDK8-mediated transcriptional reprogramming is essential for an extensive gene expression. Constitutive knockouts of the cdk8 gene are lethal at the morula stage. For modeling transcriptional reprogramming in an adult organism, we investigated the possibility to attenuate the CDK8 kinase activity with a F97G mutation in exon 3 of the cdk8 gene. According to preliminary experimental data, this mutation should lead to a decrease in CDK8 kinase activity. To edit the genome of laboratory mice, the CRISPR/Cas9 technology was used, in which the introduction of a double-stranded gap occurred at a distance of 128 nucleotide pairs from the planned site of the introduced mutation. To introduce the mutation, a matrix for homologous repair was used as part of plasmid DNA, with homologous arms 903 and 484 bp in the 5'-3' region from the point of double-stranded rupture, respectively. As a result, mice with site-specific target mutations in exon 3 of the cdk8 gene were obtained. We for the first time demonstrated a high efficacy of the mutation 128 bp apart from the site of double-strand break. Viable animals with the F97G mutation in the catalytic domain of CDK8 kinase were obtained for the first time. The resulting cdk8 mutant mice will be used in subsequent studies to simulate the processes involving transcription reprogramming.

Clp1, a polyribonucleotide 5′-hydroxyl kinase in eukaryotes, is involved in pretRNA splicing and mRNA 3′-end formation. Enzymes similar in amino acid sequence to Clp1, Nol9, and Grc3, are present in some eukaryotes and are involved in prerRNA processing. However, our knowledge of how these Clp1 family proteins evolved and diversified is limited. We conducted a large-scale molecular evolutionary analysis of the Clp1 family proteins in all living organisms for which protein sequences are available in public databases. The phylogenetic distribution and frequencies of the Clp1 family proteins were investigated in complete genomes of Bacteria, Archaea and Eukarya. In total, 3,557 Clp1 family proteins were detected in the three domains of life, Bacteria, Archaea, and Eukarya. Many were from Archaea and Eukarya, but a few were found in restricted, phylogenetically diverse bacterial species. The domain structures of the Clp1 family proteins also differed among the three domains of life. Although the proteins were, on average, 555 amino acids long (range, 196–2,728), 122 large proteins with >1,000 amino acids were detected in eukaryotes. These novel proteins contain the conserved Clp1 polynucleotide kinase domain and various other functional domains. Of these proteins, >80% were from Fungi or Protostomia. The polyribonucleotide kinase activity of Thermus scotoductus Clp1 (Ts-Clp1) was characterized experimentally. Ts-Clp1 preferentially phosphorylates single-stranded RNA oligonucleotides (Km value for ATP, 2.5 µM), or single-stranded DNA at higher enzyme concentrations. We propose a comprehensive assessment of the diversification of the Clp1 family proteins and the molecular evolution of their functional domains.

The majority of melanomas carry an oncogenic BRAF mutation (BRAF(V600E)), which results in constitutive kinase activity driving melanoma proliferation. While inhibitors of BRAF(V600E) (BRAFi) effectively lead to rapid tumor shrinkage, most patients treated with BRAFi develop acquired resistance. Identification of factors as regulators of melanoma growth and as potential sources of resistance is thus crucial for the design of improved therapies to treat advanced melanoma with more durable responses. Here, we show that KH-type splicing regulatory protein (KSRP) is critical for proliferation of melanoma cells without and with acquired resistance to vemurafenib. Silencing KSRP reduces cell proliferation and augments the growth suppressive effects of vemurafenib. We identify killin (KLLN), a p53-regulated DNA replication inhibitor, as a downstream effector of growth inhibition by KSRP silencing and demonstrate that KSRP promotes decay of KLLN mRNA through an RNA-protein interaction. Using heterologous mRNA reporters, we show that a U-rich element within the 3′ untranslated region of KLLN is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of melanoma cell growth in part through controlling KLLN mRNA stability.

The subpeduncle lobe/olfactory lobe–optic gland axis is called the endocrine regulation center of cephalopods. However, little is known about the mechanism of the subpeduncle lobe/olfactory lobe-optic gland axis regulate the sexual maturation and post-reproductive death of Sepia esculenta Hoyle. The primary objective of this study was to provide basic information for revealing the mechanism of the subpeduncle lobe/olfactory lobe–optic axis regulating the rapid post-reproductive death of S. esculenta. In this paper, Illumina sequencing based transcriptome analysis was performed on the brain tissue of female S. esculenta in the three key developmental stages: growth stage (BG), spawning stage (BS), and post-reproductive death stage (BA). A total of 66.19 Gb Illumina sequencing data were obtained. A comparative analysis of the three stages showed 2609, 3333, and 170 differentially expressed genes (DEGs) in BG-vs-BA, BG-vs-BA, and BS-vs-BA, respectively. The Gene Ontology (GO) enrichment analysis of DEGs revealed that the regulation of cyclin-dependent protein serine/threonine kinase activity, oxidative phosphorylation, and respiratory chain were significantly enriched. The significant enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway identified pathways associated with the regulation of death, such as the mammalian target of rapamycin (mTOR) signaling pathway, AMPK signaling pathway, oxidative phosphorylation, and cell cycle. The post-reproductive death of S. esculenta was found to be a complex energy steady-state regulation network system. The mTOR acted as an energy receptor and had a key role in regulating energy homeostasis.

Niemann-Pick disease, type C1 (NPC1) is an inborn error of metabolism that results in endolysosomal accumulation of unesterified cholesterol. Clinically, NPC1 manifests as cholestatic liver disease in the newborn or as a progressive neurogenerative condition characterized by cerebellar ataxia and cognitive decline. Currently there are no FDA approved therapies for NPC1. Thus, understanding the pathological processes that contribute to neurodegeneration will be important in both developing and testing potential therapeutic interventions. Neuroinflammation and necroptosis contribute to the NPC1 pathological cascade. Receptor Interacting Protein Kinase 1 and 3 (RIPK1 and RIPK3), are protein kinases that play a central role in mediating neuronal necroptosis. Our prior work suggested that pharmacological inhibition of RIPK1 had a significant but modest beneficial effect; however, the inhibitors used in that study had suboptimal pharmacokinetic properties. In this work we evaluated both pharmacological and genetic inhibition of RIPK1 kinase activity. Lifespan in both Npc1(−/−)mice treated with GSK’547, a RIPK1 inhibitor with better pharmacokinetic properties, and Npc1(−/−) :Ripk1(kd/kd) double mutant mice was significantly increased. In both cases the increase in lifespan was modest, suggesting that the therapeutic potential of RIPK1 inhibition, as a monotherapy, is limited. We thus investigated the potential of combining RIPK1 inhibition with 2-hydroxypropyl-β-cyclodextrin (HPβCD) therapy HPβCD has been shown to slow neurological disease progression in NPC1 mice, cats and patients. HPpβCD appeared to have an additive positive effect on the pathology and survival of Npc1(−/−):Ripk1(kd/kd) mice. RIPK1 and RIPK3 are both critical components of the necrosome, thus we were surprised to observe no increase survival in Npc1(−/−);Ripk3(−/−) mice compared to Npc1(−/−) mice. These data suggest that although necroptosis is occurring in NPC1, the observed effects of RIPK1 inhibition may be related to its RIPK3-independent role in neuroinflammation and cytokine production.

Recent studies have demonstrated an effect of neurotrophins, particularly brain-derived neurotrophic factor (BDNF), on airway contractility [via increased airway smooth muscle (ASM) intracellular calcium [Ca(2+)](i)] and remodeling (ASM proliferation and extracellular matrix formation) in the context of airway disease. In the present study, we examined the role of BDNF in allergen-induced airway inflammation using 2 transgenic models: 1) tropomyosin-related kinase B (TrkB) conditional knockin (TrkBKI) mice allowing for inducible, reversible disruption of BDNF receptor kinase activity by administration of 1NMPP1, a PP1 derivative, and 2) smooth muscle–specific BDNF knockout (BDNF(fl/fl)/SMMHC11(Cre/0)) mice. Adult mice were intranasally challenged with PBS or mixed allergen (Alternaria alternata, Aspergillus fumigatus, house dust mite, and ovalbumin) for 4 wk. Our data show that administration of 1NMPP1 in TrkBKI mice during the 4-wk allergen challenge blunted airway hyperresponsiveness (AHR) and reduced fibronectin mRNA expression in ASM layers but did not reduce inflammation per se. Smooth muscle–specific deletion of BDNF reduced AHR and blunted airway fibrosis but did not significantly alter airway inflammation. Together, our novel data indicate that TrkB signaling is a key modulator of AHR and that smooth muscle–derived BDNF mediates these effects during allergic airway inflammation.—Britt, R. D., Jr., Thompson, M. A., Wicher, S. A., Manlove, L. J., Roesler, A., Fang, Y.-H., Roos, C., Smith, L., Miller, J. D., Pabelick, C. M., Prakash, Y. S. Smooth muscle brain-derived neurotrophic factor contributes to airway hyperreactivity in a mouse model of allergic asthma.

For breast-fed infants, human milk is a source of various nutrients (e.g., proteins, peptides, antibodies) and bioactive components that promote neonatal growth and protect infants from viral and bacterial infection. Moreover, in terms of infant nutrition and protection the functions of many human milk components are very different from those of blood and other biological fluids of healthy adults. For example, catalytic antibodies ("abzymes") with synthetic activities (protein, oligosaccharide, and lipid kinase activities) have been found in human breast milk that are absent in the blood of healthy people. Abzymes with hydrolyzing functions have been detected not only in milk, but also in the blood of patients with autoimmune diseases. Obviously, feeding newborns human milk has a very specific role and it is a unique aspect of mammalian nutrition. Ribonuclease and DNase autoantibodies or abzymes are found in milk and blood of lactating women, but not in blood sera of healthy men and nonpregnant woman. Here, we present the first evidence that human milk secretory IgA molecules (sIgA) can effectively hydrolyze ribooligonucleotides containing 23 different bases [(pN)23 ribooligonucleotides] and 4 microRNAs: miR-9-5p, miR-219-2-3p, miR-137, and miR-219a-5p. Ribonuclease activity is an inherent property of sIgAs. We showed that 7 individual sIgAs hydrolyzed the ribooligonucleotides (pA)23, (pU)23, and (pC)23 nonspecifically and with comparable efficiency, whereas hydrolysis of the 4 microRNAs by sIgAs was site-specific. Sites of hydrolysis of 4 microRNAs by IgG from blood of patients with schizophrenia have been previously identified. The sites of hydrolysis of 4 microRNAs by sIgA-abzymes were very different from the previously identified sites of hydrolysis by IgG in patients with schizophrenia. In addition, in contrast to IgG, milk sIgAs efficiently hydrolyzed microRNAs in their loop and duplex regions.

Non-coding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs/miRs), have significant regulatory effects on a number of biological processes in myocardial ischemia/reperfusion (I/R) injury, including cell differentiation, proliferation and apoptosis. In the present study, the expression levels of lncRNAs, miRNAs and mRNAs were evaluated in a mouse model of myocardial I/R injury. The potential functions of these differentially expressed genes were then analyzed via Gene Ontology and pathway analyses. Additionally, the interactions between lncRNA-miRNA-mRNA were predicted by constructing a competing endogenous RNA regulatory network. It was found that 14,366 lncRNAs, 151 miRNAs and 9,377 mRNAs were differentially expressed in mice hearts after I/R compared with the Sham group (fold change >2; P

The transmembrane protein 33 (TMEM33) was originally identified as an endoplasmic reticulum (ER) protein that influences the tubular structure of the ER and modulates intracellular calcium homeostasis. However, the role of TMEM33 in antiviral immunity in vertebrates has not been elucidated. In this article, we demonstrate that zebrafish TMEM33 is a negative regulator of virus-triggered interferon (IFN) induction via two mechanisms: mitochondrial antiviral signaling protein (MAVS) ubiquitination and a decrease in the kinase activity of TANK binding kinase 1 (TBK1). Upon stimulation with viral components, tmem33 was remarkably upregulated in the zebrafish liver cell line. The IFNφ1 promoter (IFNφ1pro) activity and mRNA level induced by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) were significantly inhibited by TMEM33. Knockdown of TMEM33 increased host ifn transcription. Subsequently, we found that TMEM33 was colocalized in the ER and interacted with the RLR cascades, whereas MAVS was degraded by TMEM33 during the K48-linked ubiquitination. On the other hand, TMEM33 reduced the phosphorylation of mediator of IFN regulatory factor 3 (IRF3) activation (MITA)/IRF3 by acting as a decoy substrate of TBK1, which was also phosphorylated. A functional domain assay revealed that the N-terminal transmembrane domain 1 (TM1) and TM2 regions of TMEM33 were necessary for IFN suppression. Finally, TMEM33 significantly attenuated the host cellular antiviral capacity by blocking the IFN response. Taken together, our findings provide insight into the different mechanisms employed by TMEM33 in cellular IFN-mediated antiviral process., Author summary The water environment of fish enhances the chance of aquatic viral infection which leads to host IFN production, thus fish IFN as a critical antiviral role need to be modulated precisely to avoid adverse pathogenic effects, however, the IFN expression balance in fish is still unclear. In this study, we identify that zebrafish transmembrane protein 33 (TMEM33) negatively regulates IFN production by two distinct mechanisms: TMEM33 interacted with and degraded MAVS through K48-linked ubiquitination; TMEM33 acted as a decoy substrate for cellular TBK1 and reduced the phosphorylation of MITA/IRF3. Our findings reveal the role for fish TMEM33 in regulating cellular immune responses and expand our knowledge of the IFN negative regulatory mechanisms.

Background: Bacterial effector molecules are the crucial infectious agents and are sufficient to cause pathogenesis. In the present study, pathogenesis of Salmonella enteric serovar Typhi (S. Typhi) toxic proteins on the model host Caenorhabditis elegans was investigated by exploring the host regulatory proteins during infection through quantitative proteomics approach. In this regard, the host protein extract was analysed using two-dimensional gel electrophoresis (2D-GE) and differentially regulated proteins were identified using MALDI TOF/TOF/MS analysis.Results: Out of the 150 regulated proteins identified, 95 proteins were appeared to be downregulated while 55 were upregulated. Interaction network for regulated proteins was predicted using STRING tool. Most of the downregulated proteins were found to be involved in muscle contraction, locomotion, energy hydrolysis, lipid synthesis, serine/threonine kinase activity, oxidoreductase activity and protein unfolding and upregulated proteins were found to be involved in oxidative stress pathways. Hence, cellular stress generated by S. Typhi protein extract on the model host was determined using lipid peroxidation, oxidant and antioxidant assays. In addition to that the candidate proteins resulted from the host protein extract analysis were validated by Western blotting and roles of several crucial molecular players were analyzed in vivo using wild type and mutant C. elegans.Conclusions: To the best of our knowledge, this is the first study to report the protein regulation in host C. elegans during S. Typhi toxic proteins exposure which highlights the significance of p38 MAPK and JNK immune pathways. Keywords: Caenorhabditis elegans, Bacterial toxic proteins, 2D-GE, MALDI-TOF-TOF-MS, oxidative stress pathways, Western blotting, immune pathways

TheDrosophila shaggygene (sgg, GSK-3) encodes multiple protein isoforms with serine/threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. We used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. We performed proteomic analysis of the two major proteoform classes and generated mutant lines for both of these for transcriptomic and phenotypic analysis. We uncovered distinct tissue-specific localization patterns for all of the tagged proteoforms we examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively this suggests complex developmentally regulated splicing of thesggprimary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoform classes we examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of thesgglocus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. Our findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3α and GSK-3β encoded by independent vertebrate genes. Our analysis suggests that different protein isoforms generated by alternative splicing perform distinct functions.

One major factor that contributes to the virulence of Pseudomonas aeruginosa is its ability to reside and replicate unchallenged inside airway epithelial cells. The mechanism by which P. aeruginosa escapes destruction by intracellular host defense mechanisms, such as autophagy, is not known. Here, we show that the type III secretion system effector protein ExoS facilitates P. aeruginosa survival in airway epithelial cells by inhibiting autophagy in host cells. Autophagy inhibition is independent of mTOR activity, as the latter is also inhibited by ExoS, albeit by a different mechanism. Deficiency of the critical autophagy gene Atg7 in airway epithelial cells, both in vitro and in mouse models, greatly enhances the survival of ExoS-deficient P. aeruginosa but does not affect the survival of ExoS-containing bacteria. The inhibitory effect of ExoS on autophagy and mTOR depends on the activity of its ADP-ribosyltransferase domain. Inhibition of mTOR is caused by ExoS-mediated ADP ribosylation of RAS, whereas autophagy inhibition is due to the suppression of autophagic Vps34 kinase activity.

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. Here we show that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) and CryAB. NUAK biochemically and genetically interacts with Drosophila Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. We further demonstrate that Fil is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, our results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases., Author summary Non-dividing muscle and nerve cells have limited options to clear harmful biological insults. One such insult is the progressive accumulation of damaged and misfolded proteins that ultimately destroy cellular function and may result in cell or organismal death. Understanding how and why normal protein turnover occurs in healthy tissue is essential for the eventual treatment of age-related and/or degenerative diseases that result from abnormal protein accumulation. Using the large, easily manipulated muscles in fruit fly larvae, we find that loss of the evolutionarily conserved NUAK protein results in cellular degeneration due to the abnormal accumulation of certain proteins, including Fil and CryAB. Moreover, we identify Stv/BAG3 and its binding partner Hsc70-4 to be crucial for NUAK function as overexpression of Stv rescues the NUAK degenerative muscle phenotype. This work is the first to uncover NUAK as a key regulator of protein degradation through autophagy and may provide a novel therapeutic target for the treatment of protein aggregate myopathies.

Protein kinase D (PKD) controls secretion from the trans-Golgi network (TGN) by phosphorylating phosphatidylinositol 4-kinase IIIβ and proteins that bind and/or transfer phosphatidylinositol 4-phosphate (PtdIns-4P), such as oxysterol-binding protein (OSBP) and ceramide transfer protein. Here, we investigated the consequences of PKD phosphorylation of OSBP at endoplasmic reticulum (ER)-Golgi membrane contact sites (MCS). Results with OSBP phospho-mutants revealed that PKD phosphorylation did not affect sterol and PtdIns-4P binding, activation of sphingomyelin (SM) synthesis at Golgi-ER MCS or other OSBP phospho-sites. Instead, an interaction was identified between the N-terminal region of OSBP and PKD1 that was independent of kinase activity and OSBP phosphorylation status. S916 autophosphorylation of PKD1 was inhibited by OSBP expression suggesting the interaction negatively regulates PKD1 activity. Stimulation of PKD1 activity by phorbol ester promoted the Golgi-localization of wild-type and phospho-mutants of OSBP but did not affect OSBP-dependent SM synthesis. Only when wild-type or kinase-dead PKD1 was overexpressed was 25-hydroxycholesterol-activated SM synthesis inhibited. We conclude that OSBP and PKD1 form a complex that inhibits both the oxysterol-dependent activity of OSBP at the ER-Golgi and activation of PKD1. Formation of the complex was independent of PKD1 activity and phosphorylation of OSBP.

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) is a central protein in necroptosis with great potential as a target for treating necroptosis-associated diseases, such as Crohn's disease. However, blockade of RIPK3 kinase activity leads to unexpected RIPK3-initiated apoptosis. Herein, we found that PP2, a known SRC inhibitor, inhibits TNF-α-induced necroptosis without initiating apoptosis. Further investigation showed that PP2 acts as an inhibitor of not only SRC but also RIPK3. PP2 does not disturb the integrity of the RIPK1-RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL) necroptosome or the autophosphorylation of RIPK3 at T231/S232 but disrupts RIPK3 oligomerization, thereby impairing the phosphorylation and oligomerization of MLKL. These results demonstrate the essential role of RIPK3 oligomerization in necroptosis and suggest a potential RIPK3 oligomerization-targeting strategy for therapeutic development.

Targeting the interaction between PKC isoforms and their anchoring proteins can specifically regulate kinase activity. εV1-2 and pseudoεRACK peptides, derived from the PKCε C2 domain, modulate its association with receptor for activated C-kinase 2 (RACK2) and thus its function. Details of these interactions remain obscure, and we therefore investigated binding of these peptides using biophysical techniques. Surface plasmon resonance (SPR) indicated that the inhibitory εV1-2 peptide bound to RACK2, and inhibited PKCε binding as expected. In contrast, SPR and NMR demonstrated that the activating pseudoεRACK peptide and related sequences did not bind to PKCε, indicating that their mechanisms of action do not involve binding to the kinase as previously proposed. Our results clarify which interactions could be targeted in developing new therapeutics that inhibit PKCε-RACK2 interaction.

Neuronal pruning is a commonly observed phenomenon for the developing nervous systems to ensure precise wiring of neural circuits. The function of Ik2 kinase and its downstream mediator, Spindle-F (Spn-F), are essential for dendrite pruning of Drosophila sensory neurons during development. However, little is known about how Ik2/Spn-F signaling is transduced in neurons and ultimately results in dendrite pruning. Our genetic analyses and rescue experiments demonstrated that the small GTPase Rab11, especially the active GTP-bound form, is required for dendrite pruning. We also found that Rab11 shows genetic interactions with spn-F and ik2 on pruning. Live imaging of single neurons and antibody staining reveal normal Ik2 kinase activation in Rab11 mutant neurons, suggesting that Rab11 could have a functional connection downstream of and/or parallel to the Ik2 kinase signaling. Moreover, we provide biochemical evidence that both the Ik2 kinase activity and the formation of Ik2/Spn-F/Rab11 complexes are central to promote Rab11 activation in cells. Together, our studies reveal that a critical role of Ik2/Spn-F signaling in neuronal pruning is to promote Rab11 activation, which is crucial for dendrite pruning in neurons., Author summary During metamorphosis in Drosophila, both the central and peripheral nervous systems undergo substantial neuronal remodeling, such as the cell death of most larval neurons and regeneration of adult neurons, while few larval neurons remain alive and prune their branches. Pruning is a self-destruction program, and thus requires to be tightly controlled within single neurons spatially and temporally during development. Recent studies have shown a strong correlation between pruning and human psychiatric disorders, such as schizophrenia and autism. Drosophila sensory neurons that undergo dendrite pruning provide us an opportunity to study the regulatory mechanism of neuronal pruning. Previously, we identified an IKK-related kinase Ik2 that is essential and sufficient for dendrite pruning, and a coiled-coil protein Spindle-F that mediates Ik2-dependent pruning activity in neurons. However, what are the downstream targets of Ik2/Spindle-F signaling in dendrite pruning remains unclear. In this study, we found that the small GTPase Rab11, especially the active GTP-bound form, is required for dendrite pruning in neurons. We further demonstrated that both the Ik2 kinase activity and Ik2/Spindle-F complexes are essential to enhance Rab11 activation in neurons during dendrite pruning.

Interleukin 17 (IL‐17) is an important inducer of tissue inflammation and is involved in numerous autoimmune diseases. However, how its signal transduction is regulated is not well understood. Here, we report that nuclear Dbf2‐related kinase 1 (NDR1) functions as a positive regulator of IL‐17 signal transduction and IL‐17‐induced inflammation. NDR1 deficiency or knockdown inhibits the IL‐17‐induced phosphorylation of p38, ERK1/2, and p65 and the expression of chemokines and cytokines, whereas the overexpression of NDR1 promotes IL‐17‐induced signaling independent of its kinase activity. Mechanistically, NDR1 interacts with TRAF3 and prevents its binding to IL‐17R, which promotes the formation of an IL‐17R‐Act1‐TRAF6 complex and downstream signaling. Consistent with this, IL‐17‐induced inflammation is significantly reduced in NDR1‐ deficient mice, and NDR1 deficiency significantly protects mice from MOG‐induced experimental autoimmune encephalomyelitis (EAE) and 2,4,6‐trinitrobenzenesulfonic acid (TNBS)‐induced colitis likely by its inhibition of IL‐17‐mediated signaling pathway. NDR1 expression is increased in the colons of ulcerative colitis (UC) patients. Taken together, these findings suggest that NDR1 is involved in the development of autoimmune diseases. ![][1] IL‐17 induces tissue inflammation and is involved in many autoimmune reactions. This study shows that the nuclear kinase NDR1 interacts with TRAF3 and promotes the formation of IL‐17R‐Act1‐TRAF6 complexes, which trigger downstream signal transduction and inflammation. [1]: /embed/graphic-1.gif

Understanding the mechanism underlying the physiological divergence of species is a long-standing issue in evolutionary biology. The circadian clock is a highly conserved system existing in almost all organisms that regulates a wide range of physiological and behavioral events to adapt to the day-night cycle. Here, the interactions between hCK1ϵ/δ/DBT (Drosophila ortholog of CK1δ/ϵ) and serine-rich (SR) motifs from hPER2 (ortholog of Drosophila per) were reconstructed in a Drosophila circadian system. The results indicated that in Drosophila, the SR mutant form hPER2S662G does not recapitulate the mouse or human mutant phenotype. However, introducing hCK1δ (but not DBT) shortened the circadian period and restored the SR motif function. We found that hCK1δ is catalytically more efficient than DBT in phosphorylating the SR motif, which demonstrates that the evolution of CK1δ activity is required for SR motif modulation. Moreover, an abundance of phosphorylatable SR motifs and the striking emergence of putative SR motifs in vertebrate proteins were observed, which provides further evidence that the correlated evolution between kinase activity and its substrates set the stage for functional diversity in vertebrates. It is possible that such correlated evolution may serve as a biomarker associated with the adaptive benefits of diverse organisms. These results also provide a concrete example of how functional synthesis can be achieved through introducing evolutionary partners in vivo.

Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound sensorineural hearing loss locus, DFNB100 on chromosome 5q13.2-q23.2. Exome enrichment followed by massive parallel sequencing revealed a c.2510G>A transition variant in PPIP5K2 that segregated with DFNB100-associated hearing loss in two large apparently unrelated Pakistani families. PPIP5Ks enzymes interconvert 5-IP7 and IP8, two key members of the inositol pyrophosphate (PP-IP) cell-signaling family. Their actions at the interface of cell signaling and bioenergetic homeostasis can impact many biological processes. The c.2510G>A transition variant is predicted to substitute a highly invariant arginine residue with histidine (p.Arg837His) in the phosphatase domain of PPIP5K2. Biochemical studies revealed that the p.Arg837His variant reduces the phosphatase activity of PPIP5K2 and elevates its kinase activity. We found that in mouse inner ear, PPIP5K2 is expressed in the cochlear and vestibular sensory hair cells, supporting cells and spiral ganglion neurons. Mice homozygous for a targeted deletion of the Ppip5k2 phosphatase domain exhibit degeneration of cochlear outer hair cells and elevated hearing thresholds. Our demonstration that PPIP5K2 has a role in hearing in humans indicates that PP-IP signaling is important to hair cell maintenance and function within inner ear., Author summary Exome sequencing coupled with homozygosity mapping was used to identify a missense variant [c.2510G>A; p.(Arg837His)] in PPIP5K2 at the DFNB100 locus that is associated with nonsyndromic, prelingual sensorineural deafness in two large consanguineous Pakistani families. PPIP5Ks are pivotal enzymes for regulating inositol pyrophosphate (PP-IP) turnover. Biochemical analyses revealed that, compared to wild type human PPIP5K2, the PPIP5K2R837H variant exhibited lower phosphatase activity and higher kinase activity, indicating that it promotes increased metabolic flux from 5-IP7 to IP8 in vivo. In rodent inner ears, PPIP5K2 immunoreactivity was observed in the cochlear and vestibular hair cells, supporting cells, and spiral ganglion neurons. Mouse mutants homozygous for the targeted deletion of Ppip5k2 phosphatase domain exhibit degeneration of cochlear outer hair cells and progressive hearing loss. Our work provides the first description of any amino acid variant of PPIP5K2 that is both functionally-significant and associates with a human disorder. The ‘futile cycling’ of the kinase/phosphatase activity of PPIP5K2 makes inner ear function particularly susceptible to even minor changes in the phosphatase activity of PPIP5K2. We have shown that a pathogenic variant in PPIP5K2 is associated with hearing loss in humans. Thus, PPIP5K2 is given new clinical significance by our observations.

We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10-13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. However, when tested against cyclin B-dependent kinase, we found a strong inhibitory effect. Both with human Cdk1/CycB and S. pombe Cdc2/B-type cyclin the purified fraction from Superdex 200 that inhibited Dm-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3'-5'-exonuclease part of the DNA polymerase (Klenow polymerase) was activated.

Protein hydroxylation is a post-translational modification catalyzed by 2-oxoglutarate-dependent dioxygenases. The hydroxylation modification can take place on various amino acids, including but not limited to proline, lysine, asparagine, aspartate and histidine. A classical example of this modification is hypoxia inducible factor alpha (HIF-α) prolyl hydroxylation, which affects HIF-α protein stability via the Von-Hippel Lindau (VHL) tumor suppressor pathway, a Cullin 2-based E3 ligase adaptor protein frequently mutated in kidney cancer. In addition to protein stability regulation, protein hydroxylation may influence other post-translational modifications or the kinase activity of the modified protein (such as Akt and DYRK1A/B). In other cases, protein hydroxylation may alter protein-protein interaction and its downstream signaling events in vivo (such as OTUB1, MAPK6 and eEF2K). In this review, we highlight the recently identified protein hydroxylation targets and their pathophysiological roles, especially in cancer settings. Better understanding of protein hydroxylation will help identify novel therapeutic targets and their regulation mechanisms to foster development of more effective treatment strategies for various human cancers.

Recent recognition that TGF-β signaling disruption is involved in the development of aortic aneurysms has led to renewed investigations into the role of TGF-β biology in the aortic wall. We previously found that the type I receptor of TGF-β (TGFBR2) receptor contributes to formation of ascending aortic aneurysms and dissections (AADs) induced by smooth muscle cell (SMC)-specific, postnatal deletion of Tgfbr1 (Tgfbr1(iko)). Here, we aimed to decipher the mechanistic signaling pathway underlying the pathogenic effects of TGFBR2 in this context. Gene expression profiling demonstrated that Tgfbr1(iko) triggers an acute inflammatory response in developing AADs, and Tgfbr1(iko) SMCs express an inflammatory phenotype in culture. Comparative proteomics profiling and mass spectrometry revealed that Tgfbr1(iko) SMCs respond to TGF-β1 stimulation via robust up-regulation of cyclophilin A (CypA). This up-regulation is abrogated by inhibition of TGFBR2 kinase activity, small interfering RNA silencing of Tgfbr2 expression, or inhibition of SMAD3 activation. In mice, Tgfbr1(iko) rapidly promotes CypA production in SMCs of developing AADs, whereas treatment with a CypA inhibitor attenuates aortic dilation by 56% (P = 0.003) and ameliorates aneurysmal degeneration (P = 0.016). These protective effects are associated with reduced aneurysm-promoting inflammation. Collectively, these results suggest a novel mechanism, wherein loss of type I receptor of TGF-β triggers promiscuous, proinflammatory TGFBR2 signaling in SMCs, thereby promoting AAD formation.—Zhou, G., Liao, M., Wang, F., Qi, X., Yang, P., Berceli, S. A., Sharma, A. K., Upchurch, G. R., Jr., Jiang, Z. Cyclophilin A contributes to aortopathy induced by postnatal loss of smooth muscle TGFBR1.

The leucine-rich repeat kinase 2 (LRRK2) is expressed in various immune cells and involved in regulating inflammatory processes. LRRK2 facilitates calcium extrusion exchanger and sodium-calcium exchanger activity and hence influences intracellular Ca2+ concentration in dendritic cells (DCs). DC maturation and migration are governed by the intracellular Ca2+ concentration, but the related mechanisms whereby LRRK2 regulates DC function and involved Ca2+ channels are still under investigation. In the previous study, we found that LRRK2-/- DCs exhibited higher store-operated Ca2+ entry (SOCE) activity than LRRK2+/+ DCs. Herein, we ascertained the exact SOCE components by using genetic, pharmacological, and fluorescent approaches. Ca2+ imaging showed that LRRK2 kinase activity negatively modulated SOCE activity. Moreover, LRRK2 deficiency resulted in an enhanced migration capacity of DCs but had little effect on the maturation process. SOCE is widely known to regulate DC functions; we wanted to dissect the reason why LRRK2 specifically influenced DC migration and therefore silenced ORAI1, ORAI2, and ORAI3, respectively. Transwell assays showed that both ORAI1 and ORAI2 silencing markedly decreased the migration of DCs, but only ORAI1 deficiency influenced the expression of maturation markers CD11c, CD86, and major histocompatibility complex class II. Of note, LRRK2 deficiency increased ORAI2 expression but not that of ORAI1 and ORAI3. Thus, we suggest that LRRK2 modulates DC migration by interfering with ORAI2.-Yan, J., Zhao, W., Gao, C., Liu, X., Zhao, X., Wei, T., Gao, Z. Leucine-rich repeat kinase 2 regulates mouse dendritic cell migration by ORAI2.

Glucocorticoids (GCs) potently induce T-cell apoptosis in a GC receptor (GR)-dependent manner and are used to control lymphocyte function in clinical practice. However, its downstream pathways remain controversial. Here, we showed that GC-induced transcript 1 (GLCCI1) is a novel downstream molecule of the GC-GR cascade that acts as an antiapoptotic mediator in thymic T cells. GLCCI1 was highly phosphorylated and colocalized with microtubules in GLCCI1-transfected human embryonic kidney QBI293A cells. GR-dependent up-regulation of GLCCI1 was associated with GC-induced proapoptotic events in a cultured thymocyte cell line. However, GLCCI1 knockdown in a thymocyte cell line led to apoptosis. Consistently, transgenic mice overexpressing human GLCCI1 displayed enlarged thymi that consisted of larger numbers of thymocytes. Further molecular characterization showed that GLCCI1 bound to both dynein light chain LC8-type 1 (LC8) and its functional kinase, p21-protein activated kinase 1 (PAK1), thereby inhibiting the kinase activity of PAK1 toward LC8 phosphorylation, a crucial event in apoptotic signaling. GLCCI1 induction facilitated LC8 dimer formation and reduced Bim expression. Thus, GLCCI1 is a candidate factor involved in apoptosis regulation of thymic T cells.-Kiuchi, Z., Nishibori, Y., Kutsuna, S., Kotani, M., Hada, I., Kimura, T., Fukutomi, T., Fukuhara, D., Ito-Nitta, N., Kudo, A., Takata, T., Ishigaki, Y., Tomosugi, N., Tanaka, H., Matsushima, S., Ogasawara, S., Hirayama, Y., Takematsu, H., Yan, K. GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells.