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Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of-function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens.

CaMKII is an important mediator of forms of synaptic plasticity that are thought to underly learning and memory. The CaMKII mutants K42M and K42R have been used interchangeably as research tools, although some reported phenotypic differences suggest that they may differ in the extent to which they impair ATP binding. Here, we directly compared the two mutations at the high ATP concentrations that exist within cells (~4 mM). We found that both mutations equally blocked GluA1 phosphorylation in vitro and GluN2B binding within cells. Both mutations also reduced but did not completely abolish CaMKII T286 autophosphorylation in vitro or CaMKII movement to excitatory synapses in neurons. Thus, despite previously suggested differences, both mutations appear to interfere with ATP binding to the same extent.

Background Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown. Principle Findings Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner. Conclusion Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

Positive transcription elongation factor b (P-TEFb) is a RNA polymerase II carboxyl-terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in mammalian early developmental events. In this study, using immunocytochemical analyses, we found that the P-TEFb components, CDK9, Cyclin T1 and Cyclin T2 were localized to nuclear speckles, as well as in nucleolar-like bodies in pig germinal vesicle oocytes. Using nascent RNA labeling and small molecule inhibitors, we showed that inhibition of CDK9 activity abolished the transcription of GV oocytes globally. Moreover, using fluorescence in situ hybridization, in absence of CDK9 kinase activity the production of ribosomal RNAs was impaired. We also presented the evidences indicating that P-TEFb kinase activity is essential for resumption of oocyte meiosis and embryo development. Treatment with CDK9 inhibitors resulted in germinal vesicle arrest in maturing oocytes in vitro. Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when in vitro produced zygotes were treated with CDK9 inhibitors, their development beyond the 4-cell stage was impaired. In these embryos, inhibition of CDK9 abrogated global transcriptional activity and rRNA production. Collectively, our data suggested that P-TEFb kinase activity is crucial for oocyte maturation, embryo development and regulation of RNA transcription in pig.

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.

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.

Hematopoietic progenitor kinase 1 (HPK1), a hematopoietic cell-specific Ste20-related serine/threonine kinase, is a negative regulator of signal transduction in immune cells, including T cells, B cells, and dendritic cells (DCs). In mice, HPK1 deficiency subverts inhibition of the anti-tumor immune response and is associated with functional augmentation of anti-tumor T cells. We have used a potent, small molecule HPK1 inhibitor, Compound 1, to investigate the effects of pharmacological intervention of HPK1 kinase activity in immune cells. Compound 1 enhanced Th1 cytokine production in T cells and fully reverted immune suppression imposed by the prostaglandin E2(PGE2) and adenosine pathways in human T cells. Moreover, the combination of Compound 1 with pembrolizumab, a humanized monoclonal antibody against the programmed cell death protein 1 (PD-1), demonstrated a synergistic effect, resulting in enhanced interferon (IFN)-γ production. Collectively, our results suggest that blocking HPK1 kinase activity with small molecule inhibitors alone or in combination with checkpoint blockade may be an attractive approach for the immunotherapy of cancer.

Huntington’s disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. We asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer’s disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. Here, we find that htt mutations suppress axon outgrowth defects of αβ neurons in Appl mutant MB by derepressing the activity of Abl. We show that Abl is required in MB αβ neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. We find that Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth., Author summary Understanding the normal physiological roles of proteins involved in neurodegenerative diseases can provide significant insight into disease mechanisms. Drosophila offers a powerful system in which to ask these fundamental questions. Both Htt, related to Huntington’s disease, and Appl, related to Alzheimer’s disease, have well-conserved single orthologs in the fly genome. Appl has been shown to be a conserved modulator of a Wnt-PCP signaling pathway required for axon outgrowth in the mushroom body (MB) in the Drosophila brain. However, roles for Htt in fly brain development have not been reported. Unexpectedly, we found that htt mutations suppress the axon outgrowth defects of Appl mutants in the MB, indicating a link between these two neurodegenerative proteins and a cryptic role of Htt during development. Abl tyrosine kinase is a downstream effector of the Appl receptor, and we show here that Abl is also required for MB axon outgrowth. Importantly, Abl activity must be tightly regulated as evidenced by our observations that both under and overexpression of Abl result in similar axonal defects. We demonstrate that Htt is an inhibitor of Abl activity and provide evidence that the phenotypic rescue of αβ axons in Appl mutants by reducing htt is mediated by the restoration of proper levels of Abl signaling. These data, therefore, suggest that Appl and Htt act antagonistically to maintain an optimal balance of activation and inhibition of Abl, and thereby promote the growth of MB αβ axons.

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.

Herpesviral encephalitis caused by Herpes Simplex Virus 1 (HSV-1) is one of the most devastating diseases in humans. Patients present with fever, mental status changes or seizures and when untreated, sequelae can be fatal. Herpes Simplex Encephalitis (HSE) is characterized by mainly unilateral necrotizing inflammation effacing the frontal and mesiotemporal lobes with rare involvement of the brainstem. HSV-1 is hypothesized to invade the CNS via the trigeminal or olfactory nerve, but viral tropism and the exact route of infection remain unclear. Several mouse models for HSE have been developed, but they mimic natural infection only inadequately. The porcine alphaherpesvirus Pseudorabies virus (PrV) is closely related to HSV-1 and Varicella Zoster Virus (VZV). While pigs can control productive infection, it is lethal in other susceptible animals associated with severe pruritus leading to automutilation. Here, we describe the first mutant PrV establishing productive infection in mice that the animals are able to control. After intranasal inoculation with a PrV mutant lacking tegument protein pUL21 and pUS3 kinase activity (PrV-ΔUL21/US3Δkin), nearly all mice survived despite extensive infection of the central nervous system. Neuroinvasion mainly occurred along the trigeminal pathway. Whereas trigeminal first and second order neurons and autonomic ganglia were positive early after intranasal infection, PrV-specific antigen was mainly detectable in the frontal, mesiotemporal and parietal lobes at later times, accompanied by a long lasting lymphohistiocytic meningoencephalitis. Despite this extensive infection, mice showed only mild to moderate clinical signs, developed alopecic skin lesions, or remained asymptomatic. Interestingly, most mice exhibited abnormalities in behavior and activity levels including slow movements, akinesia and stargazing. In summary, clinical signs, distribution of viral antigen and inflammatory pattern show striking analogies to human encephalitis caused by HSV-1 or VZV not observed in other animal models of disease., Author summary In developed countries, more than 50% of humans are seropositive for the neurotropic Herpes Simplex Virus 1 (HSV-1) and two to four million cases of Herpes simplex encephalitis (HSE) are reported per year worldwide. Primary infection with HSV-1 takes place via the skin or the oral mucosa followed by intraaxonal retrograde spread to sensory ganglia of the peripheral nervous system where HSV-1 usually establishes latency. Further spread to the central nervous system results in HSE, a necrotizing encephalitis effacing predominantly the temporal and frontal lobes of the brain. Mice infected with HSV-1 develop encephalitis, but do not show the typical lesions and exhibit high mortality rates. Here we demonstrate that mice infected with a mutant pseudorabies virus lacking the tegument protein pUL21 and an active viral kinase pUS3 were able to survive the productive infection but developed lymphohistiocytic encephalitis with viral antigen distribution, inflammation and associated behavioral changes comparable to HSE in humans. These striking analogies offer new perspectives to study herpesviral encephalitis in a suitable animal model.

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. βPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. βPix is expressed as alternatively spliced variants, including the ubiquitous isoform βPix-a and the neuronal isoforms βPix-b and βPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of βPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from βPix neuronal isoform knockout (βPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating βPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal βPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal βPix isoforms may promote microtubule stabilization during neurite development. βPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either βPix neuronal isoform in the βPix-NIKO neurons restores Stathmin1 phosphorylation levels, with βPix-d having a greater effect than βPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via βPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that βPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal βPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.

Vessel growth integrates diverse extrinsic signals with intrinsic signaling cascades to coordinate cell migration and sprouting morphogenesis. The pro-angiogenic effects of Vascular Endothelial Growth Factor (VEGF) are carefully controlled during sprouting to generate an efficiently patterned vascular network. We identify crosstalk between VEGF signaling and that of the secreted ligand Semaphorin 3fb (Sema3fb), one of two zebrafish paralogs of mammalian Sema3F. The sema3fb gene is expressed by endothelial cells in actively sprouting vessels. Loss of sema3fb results in abnormally wide and stunted intersegmental vessel artery sprouts. Although the sprouts initiate at the correct developmental time, they have a reduced migration speed. These sprouts have persistent filopodia and abnormally spaced nuclei suggesting dysregulated control of actin assembly. sema3fb mutants show simultaneously higher expression of pro-angiogenic (VEGF receptor 2 (vegfr2) and delta-like 4 (dll4)) and anti-angiogenic (soluble VEGF receptor 1 (svegfr1)/ soluble Fms Related Receptor Tyrosine Kinase 1 (sflt1)) pathway components. We show increased phospho-ERK staining in migrating angioblasts, consistent with enhanced Vegf activity. Reducing Vegfr2 kinase activity in sema3fb mutants rescues angiogenic sprouting. Our data suggest that Sema3fb plays a critical role in promoting endothelial sprouting through modulating the VEGF signaling pathway, acting as an autocrine cue that modulates intrinsic growth factor signaling., Author summary To supply tissues with essential oxygen and nutrients, blood vessel development is carefully orchestrated by positive ‘go’ and negative ‘stop’ growth signals as well as directional cues to shape patterning. Semaphorin proteins are named after the ‘Semaphore’ railway signaling system that directed trains along the appropriate tracks. Our work highlights the role of the Semaphorin 3fb protein in providing a pro-growth signal to developing vessels. Semaphorin 3fb is both produced by, and acts on the precursors of blood vessels as they migrate, a process known as autocrine control. We find that losing Semaphorin 3fb leads to stalled blood vessel growth, indicating it normally acts as a positive signal. It acts via modulating the VEGF growth factor signaling pathway that in turn, controls the migration process. We propose that Semaphorin3b fine-tunes vessel growth, thus ensuring a properly patterned network develops.

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.

Recent evidence implicates mononuclear diploid cardiomyocytes as a proliferative and regenerative subpopulation of the postnatal heart. The number of these cardiomyocytes is a complex trait showing substantial natural variation among inbred mouse strains based on the combined influences of multiple polymorphic genes. One gene confirmed to influence this parameter is the cardiomyocyte-specific kinase Tnni3k. Here, we have studied Tnni3k alleles across a number of species. Using a newly-generated kinase-dead allele in mice, we show that Tnni3k function is dependent on its kinase activity. In an in vitro kinase assay, we show that several common human TNNI3K kinase domain variants substantially compromise kinase activity, suggesting that TNNI3K may influence human heart regenerative capacity and potentially also other aspects of human heart disease. We show that two kinase domain frameshift mutations in mice cause loss-of-function consequences by nonsense-mediated decay. We further show that the Tnni3k gene in two species of mole-rat has independently devolved into a pseudogene, presumably associated with the transition of these species to a low metabolism and hypoxic subterranean life. This may be explained by the observation that Tnni3k function in mice converges with oxidative stress to regulate mononuclear diploid cardiomyocyte frequency. Unlike other studied rodents, naked mole-rats have a surprisingly high (30%) mononuclear cardiomyocyte level but most of their mononuclear cardiomyocytes are polyploid; their mononuclear diploid cardiomyocyte level (7%) is within the known range (2–10%) of inbred mouse strains. Naked mole-rats provide further insight on a recent proposal that cardiomyocyte polyploidy is associated with evolutionary acquisition of endothermy., Author summary Embryonic cardiomyocytes have one diploid nucleus (like most cells of the body), but most adult cardiomyocytes are polyploid. Most adult cardiomyocytes are also post-mitotic and nonregenerative, and as a result, heart injury (such as from a heart attack) is followed by scarring and impaired function rather than by regeneration. A subset of cardiomyocytes in the adult heart remains mononuclear diploid, and recent evidence indicates that this subpopulation has proliferative and regenerative capacity. Our previous work in mice showed that the percentage of this cell population in the adult heart is a complex trait subject to the combined influence of a number of polymorphic genes. One gene that influences variation in this trait is a kinase gene known as Tnni3k. This study addresses the consequences of a number of Tnni3k alleles, both newly engineered in mice and naturally occurring in a number of species, including human and mole-rat, and studied at the phenotypic and biochemical level. These results provide insight into inter- and intra-species variation in the cardiomyocyte composition of the adult heart, and may be relevant to understanding heart regenerative ability in humans and across other species.

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.

Osteoclasts are large multinucleated cells responsible for bone resorption. Excessive inflammatory activation of osteoclasts leads to bony erosions, which are the hallmark of several diseases such as rheumatoid arthritis (RA). Salt-inducible kinases (SIK) constitute a subfamily of kinases comprising three members (SIK1, -2, and -3). Inhibition of SIK kinase activity induces an anti-inflammatory phenotype in macrophages. Since osteoclasts originate from precursors of macrophage origin, we hypothesized a role of SIK in osteoclastogenesis. We analyzed SIK1, -2 and -3 expression and function in osteoclast differentiation using the mouse macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMM). We show that all three SIK are expressed in fully differentiated osteoclasts and that in BMM-derived osteoclasts there is an increased expression of SIK1 and SIK3 proteins. Interestingly, the pan-SIK inhibitor HG-9-91-01 significantly inhibited osteoclastogenesis by dose dependently reducing osteoclast differentiation markers (i.e. CathepsinK, MMP-9 and TRAP) and bone resorbing activity. Analysis of the signaling pathways activated by RANKL in RAW cells showed that SIK inhibitors did not affect RANKL-induced ERK1/2, JNK, p38 or NF-κB activation, but induced a significant downregulation in c-Fos and NFATc1 protein levels, the two main transcription factors involved in the regulation of osteoclast-specific genes. Moreover, SIK inhibition partially increased the proteasome-mediated degradation of c-Fos. SIK2 and SIK3 knockout RAW cells were generated by the CRISPR/Cas9 approach. SIK2 KO and, to a lesser extent, SIK3 KO recapitulated the effect of SIK small molecule inhibitor, thus confirming the specificity of the effect of SIK inhibition on the reduction of osteoclastogenesis. Overall, our results support the notion that the SIK signaling pathway plays a significant role among the check-points controlling osteoclastogenesis. SIK kinase inhibitors could thus represent a potential novel therapy to prevent bone erosions.

Smooth muscle cells (SMCs) are key regulators of vascular disease and circulating smooth muscle progenitor cells may play important roles in vascular repair or remodelling. We developed enhanced protocols to derive smooth muscle progenitors from murine bone marrow and tested whether factors that are increased in atherosclerotic plaques, namely platelet-derived growth factor—BB (PDGF-BB) and monomeric collagen, can influence the smooth muscle specific differentiation, proliferation, and survival of mouse bone marrow-derived progenitor cells. During a 21 day period of culture, bone marrow cells underwent a marked increase in expression of the SMC markers α-SMA (1.93 ± 0.15 vs. 0.0008 ± 0.0003 (ng/ng GAPDH) at 0 d), SM22-α (1.50 ± 0.27 vs. 0.005 ± 0.001 (ng/ng GAPDH) at 0 d) and SM-MHC (0.017 ± 0.004 vs. 0.001 ± 0.001 (ng/ng GAPDH) at 0 d). Bromodeoxyuridine (BrdU) incorporation experiments showed that in early culture, the smooth muscle progenitor subpopulation could be identified by high proliferative rates prior to the expression of smooth muscle specific markers. Culture of fresh bone marrow or smooth muscle progenitor cells with PDGF-BB suppressed the expression of α-SMA and SM22-α, in a rapidly reversible manner requiring PDGF receptor kinase activity. Progenitors cultured on polymerized collagen gels demonstrated expression of SMC markers, rates of proliferation and apoptosis similar to that of cells on tissue culture plastic; in contrast, cells grown on monomeric collagen gels displayed lower SMC marker expression, lower growth rates (319 ± 36 vs. 635 ± 97 cells/mm2), and increased apoptosis (5.3 ± 1.6% vs. 1.0 ± 0.5% (Annexin 5 staining)). Our data shows that the differentiation and survival of smooth muscle progenitors are critically affected by PDGF-BB and as well as the substrate collagen structure.

Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the development of host immune responses that provide effective antitumor immunity against established ovarian cancer., Author Summary Toxoplasma gondii extensively manipulates cellular signaling pathways and host immune responses through secreted effector proteins, yet the host rapidly establishes T cell immunity to control acute infection thereby permitting survival of the host as well as survival of the parasite in latent infection. Recently, vaccination of mice bearing highly aggressive ovarian cancer with a safe nonreplicating, noncyst forming, vaccine strain of T. gondii was shown to effectively reverse tumor associated immune suppression and activate potent antitumor immunity. Using a new genetically tractable Δku80 vaccine strain of T. gondii we deleted multiple parasite secreted effector molecules to explore parasite specific mechanisms associated with the development of potent antitumor immunity. Our results demonstrate that specialized effector proteins secreted by T. gondii both before and after host cell invasion trigger and coordinately control the development of a potent antitumor response. Consequently, tracking and understanding the host cell pathways manipulated by these T. gondii secreted effector proteins can reveal fundamental mechanisms controlling immunity to infection and can also identify relevant mammalian cell mechanisms as new targets for devising more effective therapies against highly aggressive solid tumors.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors. © 2014 Minata et.

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8- /-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.

Zinc is an extremely important trace element that plays important roles in several biological processes. However, the function of zinc in meiotic division of porcine oocytes is unknown. In this study, we investigated the role of zinc during meiotic resumption in in vitro matured porcine oocytes. During meiotic division, a massive release of zinc was observed. The level of free zinc in the cytoplasm significantly increased during maturation. Depletion of zinc using N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn2+ chelator, blocked meiotic resumption in a dose dependent manner. The level of phosphorylated mitogen activated protein kinase (MAPK) and p34cdc2 kinase activity were reduced when zinc was depleted. Moreover, zinc depletion reduced the levels of phosphorylated protein kinase C (PKC) substrates in a dose dependent manner. Real-time PCR analysis showed that expression of the MAPK- and maturation promoting factor related genes C-mos, CyclinB1, and Cdc2 was downregulated following zinc depletion. Treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) increased phosphorylation of PKC substrates and MAPK and increased p34cdc2 kinase activity. This rescued the meiotic arrest, even in the presence of TPEN. Activation of PKC by PMA increased the level of zinc in the cytoplasm. These data demonstrate that zinc is required for meiotic resumption in porcine oocytes, and this appears to be regulated via a PKC related pathway.

The pathogenesis of diabetes-associated motility disorders are multifactorial and attributed to abnormalities in extrinsic and intrinsic innervation, and a decrease in the number of interstitial cells of Cajal, and nNOS expression and activity. Here we studied the effect of hyperglycemia on smooth muscle function. Using smooth muscles from the fundus of ob/ob mice and of wild type (WT) mice treated with 30 mM glucose (HG), we identified the molecular mechanism by which hyperglycemia upregulates RhoA/Rho kinase pathway and muscle contraction. RhoA expression, Rho kinase activity and muscle contraction were increased, while miR-133a expression was decreased in smooth muscle of ob/ob mice and in smooth muscle treated with HG. Intraperitoneal injections of pre-miR-133a decreased RhoA expression in WT mice and reversed the increase in RhoA expression in ob/ob mice. Intraperitoneal injections of antagomiR-133a increased RhoA expression in WT mice and augmented the increase in RhoA expression in ob/ob mice. The effect of pre-miR-133a or antagomiR-133a in vitro in smooth muscle treated with HG was similar to that obtained in vivo, suggesting that the expression of RhoA is negatively regulated by miR-133a and a decrease in miR-133a expression in diabetes causes an increase in RhoA expression. Oxidative stress (levels of reactive oxygen species and hydrogen peroxide, and expression of superoxide dismutase 1 and NADPH oxidase 4) was increased in smooth muscle of ob/ob mice and in HG-treated smooth muscle. Treatment of ob/ob mice with N-acetylcysteine (NAC) in vivo or addition of NAC in vitro to HG-treated smooth muscle reversed the effect of glucose on the expression of miR-133a and RhoA, Rho kinase activity and muscle contraction. NAC treatment also reversed the decrease in gastric emptying in ob/ob mice. We conclude that oxidative stress in diabetes causes a decrease in miR-133a expression leading to an increase in RhoA/Rho kinase pathway and muscle contraction.