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The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL2 mutations have been described, this probably only explains a subset of resistant cases. Using 2 complementary functional precision medicine techniques - BH3 profiling and high-throughput kinase activity mapping - we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL. Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Objective: To develop an interference-free and rapid method to elucidate Guanxin II (GX II)'s representative vasodilator absorbed bioactive compounds (ABCs) among enormous phytochemicals. Methods: The contents of ferulic acid, tanshinol, and hydroxysafflor yellow A (FTA) in GX II/rat serum after the oral administration of GX II (30 g/kg) were detected using ultra-performance liquid chromatography-mass spectrometry. Totally 18 rats were randomly assigned to the control group (0.9% normal saline), GX II (30 g/kg) and FTA (5, 28 and 77 mg/kg) by random number table method. Diastolic coronary flow velocity-time integral (VTI), i.e., coronary flow or coronary flow-mediated dilation (CFMD), and endothelium-intact vascular tension of isolated aortic rings were measured. After 12 h of exposure to blank medium or 0.5 mmol/L H2O2, endothelial cells (ECs) were treated with post-dose GX II of supernatant from deproteinized serum (PGSDS, 300 µL PGSDS per 1 mL of culture medium) or FTA (237, 1539, and 1510 mg/mL) for 10 min as control, H2O2, PGSDS and FTA groups. Nitric oxide (NO), vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), superoxide dismutase (SOD), malondialdehyde (MDA) and phosphorylated phosphoinositide 3 kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS) were analyzed. PGSDS was developed as a GX II proxy of ex vivo herbal crude extracts. Results: PGSDS effectively eliminates false responses caused by crude GX II preparations. When doses equaled the contents in GX II/its post-dose serum, FTA accounted for 98.17% of GX II -added CFMD and 92.99% of PGSDS-reduced vascular tension. In ECs, FTA/PGSDS was found to have significant antioxidant (lower MDA and higher SOD, P<0.01) and endothelial function-protective (lower VEGF, ET-1, P<0.01) effects. The increases in aortic relaxation, endothelial NO levels and phosphorylated PI3K/Akt/eNOS protein induced by FTA/PGSDS were markedly abolished by NG-nitro-L-arginine methyl ester (L-NA, eNOS inhibitor) and wortmannin (PI3K/AKT inhibitor), respectively, indicating an endothelium-dependent vasodilation via the PI3K/AKT-eNOS pathway (P<0.01). Conclusion: This study provides a strategy for rapidly and precisely elucidating GX II's representative in/ex vivo cardioprotective absorbed bioactive compounds (ABCs)-FTA, suggesting its potential in advancing precision ethnomedicine. [ABSTRACT FROM AUTHOR]

BACKGROUND: Clostridioides difficile infection (CDI) is a debilitating nosocomial disease. Postmenopausal women may have an increased risk of CDI, suggesting estrogen influence. Soybean products contain a representative estrogenic isoflavone, genistein. METHODS: The anti-inflammatory and antiapoptotic effects of genistein were determined using primary human cells and fresh colonic tissues. The effects of oral genistein therapy among mice and hamsters were evaluated. RESULTS: Within 10 days of CDI, female c57BL/6J mice in a standard environment (regular diet) had a 50% survival rate, while those with estrogen depletion and in an isoflavone-free environment (soy-free diet) had a 25% survival rate. Oral genistein improved their 10-day survival rate to 100% on a regular diet and 75% in an isoflavone-free environment. Genistein reduced macrophage inflammatory protein-1α (MIP-1α) secretion in fresh human colonic tissues exposed to toxins. Genistein inhibited MIP-1α secretion in primary human peripheral blood mononuclear cells, abolished apoptosis and BCL-2-associated X (BAX) expression in human colonic epithelial cells, and activated lysine-deficient protein kinase 1 (WNK1) phosphorylation in both cell types. The anti-inflammatory and antiapoptotic effects of genistein were abolished by inhibiting estrogen receptors and WNK1. CONCLUSIONS: Genistein reduces CDI disease activity by inhibiting proinflammatory cytokine expression and apoptosis via the estrogen receptor/G-protein estrogen receptor/WNK1 pathways.

The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe-kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.

Human neurodegenerative disorders often exhibit similar pathologies, suggesting a shared aetiology. Key pathological features of Parkinsons disease (PD) are also observed in other neurodegenerative diseases. Pantothenate Kinase-Associated Neurodegeneration (PKAN) is caused by mutations in the human PANK2 gene, which catalyzes the initial step of de novo CoA synthesis. Here, we show that fumble (fbl), the human PANK2 homolog in Drosophila, interacts with PINK1 genetically. fbl and PINK1 mutants display similar mitochondrial abnormalities, and overexpression of mitochondrial Fbl rescues PINK1 loss-of-function (LOF) defects. Dietary vitamin B5 derivatives effectively rescue CoA/acetyl-CoA levels and mitochondrial function, reversing the PINK1 deficiency phenotype. Mechanistically, Fbl regulates Ref(2)P (p62/SQSTM1 homolog) by acetylation to promote mitophagy, whereas PINK1 regulates fbl translation by anchoring mRNA molecules to the outer mitochondrial membrane. In conclusion, Fbl (or PANK2) acts downstream of PINK1, regulating CoA/acetyl-CoA metabolism to promote mitophagy, uncovering a potential therapeutic intervention strategy in PD treatment.

Protein kinase A (PKA) is a holoenzyme consisting of a regulatory (R)-subunit dimer and two catalytic (C)-subunits. There are two major families of C-subunits, Cα and Cβ, and four functionally nonredundant R-subunits (RIα, RIβ, RIIα, RIIβ). In addition to binding to and being regulated by the R-subunits, the C-subunits are regulated by two tail regions that each wrap around the N- and C-lobes of the kinase core. Although the C-terminal (Ct-) tail is classified as an intrinsically disordered region (IDR), the N-terminal (Nt-) tail is dominated by a strong helix that is flanked by short IDRs. In contrast to the Ct-tail, which is a conserved and highly regulated feature of all PKA, PKG, and protein kinase C protein kinase group (AGC) kinases, the Nt-tail has evolved more recently and is highly variable in vertebrates. Surprisingly and in contrast to the kinase core and the Ct-tail, the entire Nt-tail is not conserved in nonmammalian PKAs. In particular, in humans, Cβ actually represents a large family of C-subunits that are highly variable in their Nt-tail and also expressed in a highly tissue-specific manner. Although we know so much about the Cα1-subunit, we know almost nothing about these Cβ isoforms wherein Cβ2 is highly expressed in lymphocytes, and Cβ3 and Cβ4 isoforms account for ∼50% of PKA signaling in brain. Based on recent disease mutations, the Cβ proteins appear to be functionally important and nonredundant with the Cα isoforms. Imaging in retina also supports nonredundant roles for Cβ as well as isoform-specific localization to mitochondria. This represents a new frontier in PKA signaling. SIGNIFICANCE STATEMENT: How tails and adjacent domains regulate each protein kinase is a fundamental challenge for the biological community. Here we highlight how the N- and C-terminal tails of PKA (Nt-tails/Ct-tails) affect the structure and regulate the function of the kinase core and show the combinatorial variations that are introduced into the Nt-tail of the Cα- and Cβ-subunits in contrast to the Ct-tail, which is conserved across the entire AGC subfamily of protein kinases.

Dynamic change of mitochondrial morphology and distribution along neuronal branches are essential for neural circuitry formation and synaptic efficacy. However, the underlying mechanism remains elusive. We show here that Pink1 knockout (KO) mice display defective dendritic spine maturation, reduced axonal synaptic vesicles, abnormal synaptic connection, and attenuated long-term synaptic potentiation (LTP). Drp1 activation via S616 phosphorylation rescues deficits of spine maturation in Pink1 KO neurons. Notably, mice harboring a knockin (KI) phosphor-null Drp1S616A recapitulate spine immaturity and synaptic abnormality identified in Pink1 KO mice. Chemical LTP (cLTP) induces Drp1S616 phosphorylation in a PINK1-dependent manner. Moreover, phosphor-mimetic Drp1S616D restores reduced dendritic spine localization of mitochondria in Pink1 KO neurons. Together, this study provides the first in vivo evidence of functional regulation of Drp1 by phosphorylation and suggests that PINK1-Drp1S616 phosphorylation coupling is essential for convergence between mitochondrial dynamics and neural circuitry formation and refinement.

Coordinating the balance between development and stress responses is critical for organismal survival. However, the cellular signaling controlling this mechanism is not well understood. In Caenorhabditis elegans, it has been hypothesized that a genetic network regulated by NIPI-3/Tibbles may control the balance between animal development and immune response. Using a nipi-3(0) lethality suppressor screen in C. elegans, we reveal a novel role for N-terminal acetyltransferase C complex natc-1/2/3 and histone deacetylase hda-4, in the control of animal development. These signaling proteins act, at least in part, through a PMK-1 p38 MAP kinase pathway (TIR-1-NSY-1-SEK-1-PMK-1), which plays a critical role in the innate immunity against infection. Additionally, using a transcriptional reporter of SEK-1, a signaling molecule within this p38 MAP kinase system that acts directly downstream of C/EBP bZip transcription factor CEBP-1, we find unexpected positive control of sek-1 transcription by SEK-1 along with several other p38 MAP kinase pathway components. Together, these data demonstrate a role for NIPI-3 regulators in animal development, operating, at least in part through a PMK-1 p38 MAPK pathway. Because the C. elegans p38 MAP kinase pathway is well known for its role in cellular stress responses, the novel biological components and mechanisms pertaining to development identified here may also contribute to the balance between stress response and development.

Retrotransposons mediate gene regulation in important developmental and pathological processes. Here, we characterized the transient retrotransposon induction during preimplantation development of eight mammals. Induced retrotransposons exhibit similar preimplantation profiles across species, conferring gene regulatory activities, particularly through long terminal repeat (LTR) retrotransposon promoters. A mouse-specific MT2B2 retrotransposon promoter generates an N-terminally truncated Cdk2ap1ΔN that peaks in preimplantation embryos and promotes proliferation. In contrast, the canonical Cdk2ap1 peaks in mid-gestation and represses cell proliferation. This MT2B2 promoter, whose deletion abolishes Cdk2ap1ΔN production, reduces cell proliferation and impairs embryo implantation, is developmentally essential. Intriguingly, Cdk2ap1ΔN is evolutionarily conserved in sequence and function yet is driven by different promoters across mammals. The distinct preimplantation Cdk2ap1ΔN expression in each mammalian species correlates with the duration of its preimplantation development. Hence, species-specific transposon promoters can yield evolutionarily conserved, alternative protein isoforms, bestowing them with new functions and species-specific expression to govern essential biological divergence.

The unfolded protein response (UPR), crucial for the maintenance of endoplasmic reticulum (ER) homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here, we show that Candida albicans relies on an ER-resident protein, inositol-requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1-mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence-related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin-induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1-mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

It is difficult to imagine where the signaling community would be today without the Protein Data Bank. This visionary resource, established in the 1970s, has been an essential partner for sharing information between academics and industry for over 3 decades. We describe here the history of our journey with the protein kinases using cAMP-dependent protein kinase as a prototype. We summarize what we have learned since the first structure, published in 1991, why our journey is still ongoing, and why it has been essential to share our structural information. For regulation of kinase activity, we focus on the cAMP-binding protein kinase regulatory subunits. By exploring full-length macromolecular complexes, we discovered not only allostery but also an essential motif originally attributed to crystal packing. Massive genomic data on disease mutations allows us to now revisit crystal packing as a treasure chest of possible protein:protein interfaces where the biological significance and disease relevance can be validated. It provides a new window into exploring dynamic intrinsically disordered regions that previously were deleted, ignored, or attributed to crystal packing. Merging of crystallography with cryo-electron microscopy, cryo-electron tomography, NMR, and millisecond molecular dynamics simulations is opening a new world for the signaling community where those structure coordinates, deposited in the Protein Data Bank, are just a starting point!

Loss of the tumor suppressor NF1 leads to activation of RAS effector pathways, which are therapeutically targeted by inhibition of mTOR (mTORi) or MEK (MEKi). However, therapeutic inhibition of RAS effectors leads to the development of drug resistance and ultimately disease progression. To investigate molecular signatures in the context of NF1 loss and subsequent acquired drug resistance, we analyzed the exomes, transcriptomes, and kinomes of Nf1-mutant mouse tumor cell lines and derivatives of these lines that acquired resistance to either MEKi or mTORi. Biochemical comparisons of this unique panel of tumor cells, all of which arose in Nf1+/- mice, indicate that loss of heterozygosity of Nf1 as an initial genetic event does not confer a common biochemical signature or response to kinase inhibition. Although acquired drug resistance by Nf1-mutant tumor cells was accompanied by altered kinomes and irreversibly altered transcriptomes, functionally in multiple Nf1-mutant tumor cell lines, MEKi resistance was a stable phenotype, in contrast to mTORi resistance, which was reversible. Collectively, these findings demonstrate that Nf1-mutant tumors represent a heterogeneous group biochemically and undergo broader remodeling of kinome activity and gene expression in response to targeted kinase inhibition.

PurposeTo elucidate the molecular events in solute carrier family 4 member 11 (SLC4A11)-deficient corneal endothelium that lead to the endothelial dysfunction that characterizes the dystrophies associated with SLC4A11 mutations, congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy 4.MethodsComparative transcriptomic analysis (CTA) was performed in primary human corneal endothelial cells (pHCEnC) and murine corneal endothelial cells (MCEnC) with normal and reduced levels of SLC4A11 (SLC4A11 KD pHCEnC) and Slc4a11 (Slc4a11-/- MCEnC), respectively. Validation of differentially expressed genes was performed using immunofluorescence staining of CHED corneal endothelium, as well as western blot and quantitative PCR analysis of SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Functional analyses were performed to investigate potential functional changes associated with the observed transcriptomic alterations.ResultsCTA revealed inhibition of cell metabolism and ion transport function as well as mitochondrial dysfunction, leading to reduced adenosine triphosphate (ATP) production, in SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Co-localization of SNARE protein STX17 with mitochondria marker COX4 was observed in CHED corneal endothelium, as was activation of AMPK-p53/ULK1 in both SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC, providing additional evidence of mitochondrial dysfunction and mitophagy. Reduced Na+-dependent HCO3- transport activity and altered NH4Cl-induced membrane potential changes were observed in Slc4a11-/- MCEnC.ConclusionsReduced steady-state ATP levels and subsequent activation of the AMPK-p53 pathway provide a link between the metabolic functional deficit and transcriptome alterations, as well as evidence of insufficient ATP to maintain the Na+/K+-ATPase corneal endothelial pump as the cause of the edema that characterizes SLC4A11-associated corneal endothelial dystrophies.

Mitochondria are important sources of energy, but they are also the target of cellular stress, toxin exposure, and aging-related injury. Persistent accumulation of damaged mitochondria has been implicated in many neurodegenerative diseases. One highly conserved mechanism to clear damaged mitochondria involves the E3 ubiquitin ligase Parkin and PTEN-induced kinase 1 (PINK1), which cooperatively initiate the process called mitophagy that identifies and eliminates damaged mitochondria through the autophagosome and lysosome pathways. Parkin is a mostly cytosolic protein, but is rapidly recruited to damaged mitochondria and target them for mitophagy. Moreover, Parkin interactomes also involve signaling pathways and transcriptional machinery critical for survival and cell death. However, the mechanism that regulates Parkin protein level remains poorly understood. Here, we show that the loss of homeodomain interacting protein kinase 2 (HIPK2) in neurons and mouse embryonic fibroblasts (MEFs) has a broad protective effect from cell death induced by mitochondrial toxins. The mechanism by which Hipk2-/- neurons and MEFs are more resistant to mitochondrial toxins is in part due to the role of HIPK2 and its kinase activity in promoting Parkin degradation via the proteasome-mediated mechanism. The loss of HIPK2 leads to higher cytosolic Parkin protein levels at basal conditions and upon exposure to mitochondrial toxins, which protects mitochondria from toxin-induced damage. In addition, Hipk2-/- neurons and MEFs show increased expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1), a Parkin downstream target that can provide additional benefits via transcriptional activation of mitochondrial genes. Together, these results reveal a previously unrecognized avenue to target HIPK2 in neuroprotection via the Parkin-mediated pathway.SIGNIFICANCE STATEMENT In this study, we provide evidence that homeodomain interacting protein kinase 2 (HIPK2) and its kinase activity promote Parkin degradation via the proteasome-mediated pathway. The loss of HIPK2 increases cytosolic and mitochondrial Parkin protein levels under basal conditions and upon exposure to mitochondrial toxins, which protect mitochondria from toxin-induced damage. In addition, Hipk2-/- neurons and mouse embryonic fibroblasts also show increased expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1), a Parkin downstream target that can provide additional benefits via transcriptional activation of mitochondrial genes. These results indicate that targeting HIPK2 and its kinase activity can have neuroprotective effects by elevating Parkin protein levels.

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction.

Recent studies implicate a strong association between elevated plasma branched-chain amino acids (BCAAs) and insulin resistance (IR). However, a causal relationship and whether interrupted BCAA homeostasis can serve as a therapeutic target for diabetes remain to be established experimentally. In this study, unbiased integrative pathway analyses identified a unique genetic link between obesity-associated IR and BCAA catabolic gene expression at the pathway level in human and mouse populations. In genetically obese (ob/ob) mice, rate-limiting branched-chain α-keto acid (BCKA) dehydrogenase deficiency (i.e., BCAA and BCKA accumulation), a metabolic feature, accompanied the systemic suppression of BCAA catabolic genes. Restoring BCAA catabolic flux with a pharmacological inhibitor of BCKA dehydrogenase kinase (BCKDK) ( a suppressor of BCKA dehydrogenase) reduced the abundance of BCAA and BCKA and markedly attenuated IR in ob/ob mice. Similar outcomes were achieved by reducing protein (and thus BCAA) intake, whereas increasing BCAA intake did the opposite; this corroborates the pathogenic roles of BCAAs and BCKAs in IR in ob/ob mice. Like BCAAs, BCKAs also suppressed insulin signaling via activation of mammalian target of rapamycin complex 1. Finally, the small-molecule BCKDK inhibitor significantly attenuated IR in high-fat diet-induced obese mice. Collectively, these data demonstrate a pivotal causal role of a BCAA catabolic defect and elevated abundance of BCAAs and BCKAs in obesity-associated IR and provide proof-of-concept evidence for the therapeutic validity of manipulating BCAA metabolism for treating diabetes.

Pseudokinases are members of the protein kinase superfamily but signal primarily through noncatalytic mechanisms. Many pseudokinases contribute to the pathologies of human diseases, yet they remain largely unexplored as drug targets owing to challenges associated with modulation of their biological functions. Our understanding of the structure and physiological roles of pseudokinases has improved substantially over the past decade, revealing intriguing similarities between pseudokinases and their catalytically active counterparts. Pseudokinases often adopt conformations that are analogous to those seen in catalytically active kinases and, in some cases, can also bind metal cations and/or nucleotides. Several clinically approved kinase inhibitors have been shown to influence the noncatalytic functions of active kinases, providing hope that similar properties in pseudokinases could be pharmacologically regulated. In this Review, we discuss known roles of pseudokinases in disease, their unique structural features and the progress that has been made towards developing pseudokinase-directed therapeutics.

Ampk is an energy gatekeeper that responds to decreases in ATP by inhibiting energy-consuming anabolic processes and promoting energy-generating catabolic processes. Recently, we showed that Lkb1, an understudied kinase in B lymphocytes and a major upstream kinase for Ampk, had critical and unexpected roles in activating naïve B cells and in germinal center formation. Therefore, we examined whether Lkb1 activities during B cell activation depend on Ampk and report surprising Ampk activation with in vitro B cell stimulation in the absence of energy stress, coupled to rapid biomass accumulation. Despite Ampk activation and a controlling role for Lkb1 in B cell activation, Ampk knockout did not significantly affect B cell activation, differentiation, nutrient dynamics, gene expression, or humoral immune responses. Instead, Ampk loss specifically repressed the transcriptional expression of IgD and its regulator, Zfp318. Results also reveal that early activation of Ampk by phenformin treatment impairs germinal center formation but does not significantly alter antibody responses. Combined, the data show an unexpectedly specific role for Ampk in the regulation of IgD expression during B cell activation.

Loss of skeletal muscle mass and function is a hallmark of aging. This phenomenon has been related to a dysregulation of mitochondrial function and proteostasis. Calorie restriction (CR) has been demonstrated to delay aging and preserve function until late in life, particularly in muscle. Recently, we reported the type of dietary fat plays an important role in determining life span extension with 40% CR in male mice. In these conditions, lard fed mice showed an increased longevity compared to mice fed soybean or fish oils. In this article, we analyze the effect of 40% CR on muscle mitochondrial mass, autophagy, and mitochondrial dynamics markers in mice fed these diets. In CR fed animals, lard preserved muscle fibers structure, mitochondrial ultrastructure, and fission/fusion dynamics and autophagy, not only compared to control animals, but also compared with CR mice fed soybean and fish oils as dietary fat. We focus our discussion on dietary fatty acid saturation degree as an essential predictor of life span extension in CR mice.

The piRNA pathway is an adaptive mechanism that maintains genome stability by repression of selfish genomic elements. In the male germline of Drosophila melanogaster repression of Stellate genes by piRNAs generated from Supressor of Stellate (Su(Ste)) locus is required for male fertility, but both Su(Ste) piRNAs and their targets are absent in other Drosophila species. We found that D. melanogaster genome contains multiple X-linked non-coding genomic repeats that have sequence similarity to the protein-coding host gene vasa. In the male germline, these vasa-related AT-chX repeats produce abundant piRNAs that are antisense to vasa; however, vasa mRNA escapes silencing due to imperfect complementarity to AT-chX piRNAs. Unexpectedly, we discovered AT-chX piRNAs target vasa of Drosophila mauritiana in the testes of interspecies hybrids. In the majority of hybrid flies, the testes were strongly reduced in size and germline content. A minority of hybrids maintained wild-type array of premeiotic germ cells in the testes, but in them harmful Stellate genes were derepressed due to the absence of Su(Ste) piRNAs, and meiotic failures were observed. Thus, the piRNA pathway contributes to reproductive isolation between D. melanogaster and closely related species, causing hybrid male sterility via misregulation of two different host protein factors.

Cigarette smoke (CS)-induced accumulation of mitochondrial damage has been widely implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. Mitophagy plays a crucial role in eliminating damaged mitochondria, and is governed by the PINK1 (PTEN induced putative protein kinase 1)-PRKN (parkin RBR E3 ubiquitin protein ligase) pathway. Although both increased PINK1 and reduced PRKN have been implicated in COPD pathogenesis in association with mitophagy, there are conflicting reports for the role of mitophagy in COPD progression. To clarify the involvement of PRKN-regulated mitophagy in COPD pathogenesis, prkn knockout (KO) mouse models were used. To illuminate how PINK1 and PRKN regulate mitophagy in relation to CS-induced mitochondrial damage and cellular senescence, overexpression and knockdown experiments were performed in airway epithelial cells (AEC). In comparison to wild-type mice, prkn KO mice demonstrated enhanced airway wall thickening with emphysematous changes following CS exposure. AEC in CS-exposed prkn KO mice showed accumulation of damaged mitochondria and increased oxidative modifications accompanied by accelerated cellular senescence. In vitro experiments showed PRKN overexpression was sufficient to induce mitophagy during CSE exposure even in the setting of reduced PINK1 protein levels, resulting in attenuation of mitochondrial ROS production and cellular senescence. Conversely PINK1 overexpression failed to recover impaired mitophagy caused by PRKN knockdown, indicating that PRKN protein levels can be the rate-limiting factor in PINK1-PRKN-mediated mitophagy during CSE exposure. These results suggest that PRKN levels may play a pivotal role in COPD pathogenesis by regulating mitophagy, suggesting that PRKN induction could mitigate the progression of COPD. Abbreviations: AD: Alzheimer disease; AEC: airway epithelial cells; BALF: bronchoalveolar lavage fluid; AKT: AKT serine/threonine kinase; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; COPD: chronic obstructive pulmonary disease; CS: cigarette smoke; CSE: CS extract; CXCL1: C-X-C motif chemokine ligand 1; CXCL8: C-X-C motif chemokine ligand 8; HBEC: human bronchial epithelial cells; 4-HNE: 4-hydroxynonenal; IL: interleukin; KO: knockout; LF: lung fibroblasts; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; 8-OHdG: 8-hydroxy-2'-deoxyguanosine; OPTN: optineurin; PRKN: parkin RBR E3 ubiquitin protein ligase; PCD: programmed cell death; PFD: pirfenidone; PIK3C: phosphatidylinositol-4:5-bisphosphate 3-kinase catalytic subunit; PINK1: PTEN induced putative kinase 1; PTEN: phosphatase and tensin homolog; RA: rheumatoid arthritis; ROS: reactive oxygen species; SA-GLB1/β-Gal: senescence-associated-galactosidase, beta 1; SASP: senescence-associated secretory phenotype; SNP: single nucleotide polymorphism; TNF: tumor necrosis factor.

OBJECTIVE:Diabetic sensorimotor polyneuropathy (DSPN) affects approximately half of diabetic patients leading to significant morbidity. There is impaired neurotrophic growth factor signaling, AMP-activated protein kinase (AMPK) activity and mitochondrial function in dorsal root ganglia (DRG) of animal models of type 1 and type 2 diabetes. We hypothesized that sub-optimal insulin-like growth factor 1 (IGF-1) signaling in diabetes drives loss of AMPK activity and mitochondrial function, both contributing to development of DSPN. METHODS:Age-matched control Sprague-Dawley rats and streptozotocin (STZ)-induced type 1 diabetic rats with/without IGF-1 therapy were used for in vivo studies. For in vitro studies, DRG neurons from control and STZ-diabetic rats were cultured and treated with/without IGF-1 in the presence or absence of inhibitors or siRNAs. RESULTS:Dysregulation of mRNAs for IGF-1, AMPKα2, ATP5a1 (subunit of ATPase), and PGC-1β occurred in DRG of diabetic vs. control rats. IGF-1 up-regulated mRNA levels of these genes in cultured DRGs from control or diabetic rats. IGF-1 treatment of DRG cultures significantly (P

BackgroundInflammation is associated with cardiac remodeling and heart failure, but how it is initiated in response to nonischemic interventions in the absence of cell death is not known. We tested the hypothesis that activation of Ca2+/calmodulin-dependent protein kinase II δ (CaMKIIδ) in cardiomyocytes (CMs) in response to pressure overload elicits inflammatory responses leading to adverse remodeling.MethodsMice in which CaMKIIδ was selectively deleted from CMs (cardiac-specific knockout [CKO]) and floxed control mice were subjected to transverse aortic constriction (TAC). The effects of CM-specific CaMKIIδ deletion on inflammatory gene expression, inflammasome activation, macrophage accumulation, and fibrosis were assessed by quantitative polymerase chain reaction, histochemistry, and ventricular remodeling by echocardiography.ResultsTAC induced increases in cardiac mRNA levels for proinflammatory chemokines and cytokines in ≤3 days, and these responses were significantly blunted when CM CaMKIIδ was deleted. Apoptotic and necrotic cell death were absent at this time. CMs isolated from TAC hearts mirrored these robust increases in gene expression, which were markedly attenuated in CKO. Priming and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome, assessed by measuring interleukin-1β and NOD-like receptor pyrin domain-containing protein 3 mRNA levels, caspase-1 activity, and interleukin-18 cleavage, were increased at day 3 after TAC in control hearts and in CMs isolated from these hearts. These responses were dependent on CaMKIIδ and associated with activation of Nuclear Factor-kappa B and reactive oxygen species. Accumulation of macrophages observed at days 7 to 14 after TAC was diminished in CKO and, by blocking Monocyte Chemotactic Protein-1 signaling, deletion of CM Monocyte Chemotactic Protein-1 or inhibition of inflammasome activation. Fibrosis was also attenuated by these interventions and in the CKO heart. Ventricular dilation and contractile dysfunction observed at day 42 after TAC were diminished in the CKO. Inhibition of CaMKII, Nuclear Factor-kappa B, inflammasome, or Monocyte Chemotactic Protein-1 signaling in the first 1 or 2 weeks after TAC decreased remodeling, but inhibition of CaMKII after 2 weeks did not.ConclusionsActivation of CaMKIIδ in response to pressure overload triggers inflammatory gene expression and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome in CMs. These responses provide signals for macrophage recruitment, fibrosis, and myocardial dysfunction in the heart. Our work suggests the importance of targeting early inflammatory responses induced by CM CaMKIIδ signaling to prevent progression to heart failure.

In order to investigate the effects of clopidogrel rehabilitation on the levels of cardiac protein kinase C (PKC), cardiac heat shock protein 70 (HSP70), and cardiac index (CI) in rats with myocardial ischemia-reperfusion injury (MIRI), sixty Wistar rats are randomly divided into three groups (sham operation group, model group, and clopidogrel group), with 20 rats in each group. The clopidogrel group is given clopidogrel by gavage, and the sham operation group and the model group are given the same amount of normal saline by gavage. The experimental results show that compared with the model group, the clopidogrel group has clear horizontal lines and cell edema. The myocardial infarction rate, creatine kinase-MB (CK-MB), and malondialdehyde (MDA) of the model group and clopidogrel group in the control sham operation group significantly increase. [ABSTRACT FROM AUTHOR]

Background: Adipose tissue-derived adipokines are involved in various crosstalk between adipose tissue and other organs. Omentin1, a novel adipokine, exerts vital roles in the maintenance of body metabolism, insulin resistance and the like. However, the protective effect of omentin1 in myocardial ischemia (MI)-induced heart failure (HF) and its specific mechanism remains unclear and to be elucidated.Methods: The model of MI-induced HF mice and oxygen glucose deprivation (OGD)-injured cardiomyocytes were performed. Mice with overexpression of omentin1 were constructed by a fat-specific adeno-associated virus (AAV) vector system.Results: We demonstrated that circulating omentin1 level diminished in HF patients compared with healthy subjects. Furthermore, the fat-specific overexpression of omentin1 ameliorated cardiac function, cardiac hypertrophy, infarct size and cardiac pathological features, and also enhanced SIRT3/FOXO3a signaling in HF mice. Additionally, administration with AAV-omentin1 increased mitochondrial fusion and decreased mitochondrial fission in HF mice, as evidenced by up-regulated expression of Mfn2 and OPA1, and downregulation of p-Drp1(Ser616). Then, it also promoted PINK1/Parkin-dependent mitophagy. Simultaneously, treatment with recombinant omentin1 strengthened OGD-injured cardiomyocyte viability, restrained LDH release, and enhanced the mitochondrial accumulation of SIRT3 and nucleus transduction of FOXO3a. Besides, omentin1 also ameliorated unbalanced mitochondrial fusion-fission dynamics and activated mitophagy, thereby, improving the damaged mitochondria morphology and controlling mitochondrial quality in OGD-injured cardiomyocytes. Interestingly, SIRT3 played an important role in the improvement effects of omentin1 on mitochondrial function, unbalanced mitochondrial fusion-fission dynamics and mitophagy.Conclusion: Omentin1 improves MI-induced HF and myocardial injury by maintaining mitochondrial dynamical homeostasis and activating mitophagy via upregulation of SIRT3/FOXO3a signaling. This study provides evidence for further application of omentin1 in cardiovascular diseases from the perspective of crosstalk between heart and adipose tissue. [ABSTRACT FROM AUTHOR]

Objectives: To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1, is an autoinflammatory disease.Methods: This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling.Results: The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow.Conclusion: ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy. [ABSTRACT FROM AUTHOR]

Although aberrant EGFR signaling is widespread in cancer, EGFR inhibition is effective only in a subset of non-small cell lung cancer (NSCLC) with EGFR activating mutations. A majority of NSCLCs express EGFR wild type (EGFRwt) and do not respond to EGFR inhibition. TNF is a major mediator of inflammation-induced cancer. We find that a rapid increase in TNF level is a universal adaptive response to EGFR inhibition in NSCLC, regardless of EGFR status. EGFR signaling actively suppresses TNF mRNA levels by inducing expression of miR-21, resulting in decreased TNF mRNA stability. Conversely, EGFR inhibition results in loss of miR-21 and increased TNF mRNA stability. In addition, TNF-induced NF-κB activation leads to increased TNF transcription in a feed-forward loop. Inhibition of TNF signaling renders EGFRwt-expressing NSCLC cell lines and an EGFRwt patient-derived xenograft (PDX) model highly sensitive to EGFR inhibition. In EGFR-mutant oncogene-addicted cells, blocking TNF enhances the effectiveness of EGFR inhibition. EGFR plus TNF inhibition is also effective in NSCLC with acquired resistance to EGFR inhibition. We suggest concomitant EGFR and TNF inhibition as a potentially new treatment approach that could be beneficial for a majority of lung cancer patients.

Neuropathy is a common complication of long-term diabetes. Proposed mechanisms of neuronal damage caused by diabetes that are downstream of hyperglycemia and/or loss of insulin signaling include ischemic hypoxia, inflammation and loss of neurotrophic support. The curcumin derivative J147 is a potent neurogenic and neuroprotective drug candidate initially developed for the treatment of neurodegenerative conditions associated with aging that impacts many pathways implicated in the pathogenesis of diabetic neuropathy. Here, we demonstrate efficacy of J147 in ameliorating multiple indices of neuropathy in the streptozotocin-induced mouse model of type 1 diabetes. Diabetes was determined by blood glucose, HbA1c, and insulin levels and efficacy of J147 by behavioral, physiologic, biochemical, proteomic, and transcriptomic assays. Biological efficacy of systemic J147 treatment was confirmed by its capacity to decrease TNFα pathway activation and several other markers of neuroinflammation in the CNS. Chronic oral treatment with J147 protected the sciatic nerve from progressive diabetes-induced slowing of large myelinated fiber conduction velocity while single doses of J147 rapidly and transiently reversed established touch-evoked allodynia. Conduction slowing and allodynia are clinically relevant markers of early diabetic neuropathy and neuropathic pain, respectively. RNA expression profiling suggests that one of the pathways by which J147 imparts its protection against diabetic induced neuropathy may be through activation of the AMP kinase pathway. The diverse biological and therapeutic effects of J147 suggest it as an alternative to the polypharmaceutical approaches required to treat the multiple pathogenic mechanisms that contribute to diabetic neuropathy.

The N-methyl-d-aspartate-type glutamate receptor (NMDAR)-associated multiprotein complexes are indispensable for synaptic plasticity and cognitive functions. While purification and proteomic analyses of these signaling complexes have been performed in adult rodent and human brain, much less is known about the protein composition of NMDAR complexes in the developing brain and their modifications by neonatal hypoxic-ischemic (HI) brain injury. In this study, the postsynaptic density proteins were prepared from postnatal day 9 naïve, sham-operated and HI-injured mouse cortex. The GluN2B-containing NMDAR complexes were purified by immunoprecipitation with a mouse GluN2B antibody and subjected to mass spectrometry analysis for determination of the GluN2B binding partners. A total of 71 proteins of different functional categories were identified from the naïve animals as native GluN2B-interacting partners in the developing mouse brain. Neonatal HI reshaped the postsynaptic GluN2B interactome by recruiting new proteins, including multiple kinases, into the complexes; and modifying the existing associations within 1h of reperfusion. The early responses of postsynaptic NMDAR complexes and their related signaling networks may contribute to molecular processes leading to cell survival or death, brain damage and/or neurological disorders in term infants with neonatal encephalopathy.

Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that are critical to the intracellular replicative cycle of T. gondii including secretion of adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents that complement this expanded binding pocket. Here we explored structure-activity relationships of a series of pyrazolopyrimidine inhibitors of CDPK1 with the goal of increasing selectivity over host enzymes, improving antiparasite potency, and improving metabolic stability. The resulting lead compound 24 exhibited excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited parasite growth in vitro. Compound 24 was also effective at treating acute toxoplasmosis in the mouse, reducing dissemination to the central nervous system, and decreasing reactivation of chronic infection in severely immunocompromised mice. These findings provide proof of concept for the development of small molecule inhibitors of CDPK1 for treatment of CNS toxoplasmosis.

One of the major biological functions of the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process resembling phagocytosis. RPE phagocytosis helps maintain the viability of photoreceptors that otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. The regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration. Here, we present an integrated transcriptomic, proteomic, and phosphoproteomic analysis of phagocytosing RPE cells, utilizing three different experimental models: the human-derived RPE-like cell line ARPE-19, cultured murine primary RPE cells, and RPE samples from live mice. Our combined results indicated that early stages of phagocytosis in the RPE are mainly characterized by pronounced changes in the protein phosphorylation level. Global phosphoprotein enrichment analysis revealed involvement of PI3K/Akt, mechanistic target of rapamycin (mTOR), and MEK/ERK pathways in the regulation of RPE phagocytosis, confirmed by immunoblot analyses and in vitro phagocytosis assays. Most strikingly, phagocytosis of POS by cultured RPE cells was almost completely blocked by pharmacological inhibition of phosphorylation of Akt. Our findings, along with those of previous studies, indicate that these phosphorylation events allow the RPE to integrate multiple signals instigated by shed POS at different stages of the phagocytic process.

IntroductionOrgan-specific autoimmune diseases are believed to result from immune responses generated against self-antigens specific to each organ. However, when such responses target antigens expressed promiscuously in multiple tissues, then the immune-mediated damage may be wide spread.MethodsIn this report, we describe a mitochondrial protein, branched chain α-ketoacid dehydrogenase kinase (BCKDk ) that can act as a target autoantigen in the development of autoimmune inflammatory reactions in both heart and liver.ResultsWe demonstrate that BCKDk protein contains at least nine immunodominant epitopes, three of which, BCKDk 71-90, BCKDk 111-130 and BCKDk 141-160, were found to induce varying degrees of myocarditis in immunized mice. One of these, BCKDk 111-130, could also induce hepatitis without affecting lungs, kidneys, skeletal muscles, and brain. In immunogenicity testing, all three peptides induced antigen-specific T cell responses, as verified by proliferation assay and/or major histocompatibility complex class II/IAk dextramer staining. Finally, the disease-inducing abilities of BCKDk peptides were correlated with the production of interferon-γ, and the activated T cells could transfer disease to naive recipients.ConclusionsThe disease induced by BCKDk peptides could serve as a useful model to study the autoimmune events of inflammatory heart and liver diseases.

Liver Kinase B1 (LKB1) is known as a master kinase for 14 kinases related to the adenosine monophosphate-activated protein kinase. Two of them salt inducible kinase 3 and adenosine monophosphate-activated protein kinase a have previously been implicated in sleep regulation. We generated loss-of-function mutants for Lkb1 in both Drosophila and mice. Sleep, but not circadian rhythms, was reduced in Lkb1-mutant flies and in flies with neuronal deletion of Lkb1. Genetic interactions between Lkb1 and threonine to alanine mutation at residue 184 of adenosine monophosphate-activated protein kinase in Drosophila sleep or those between Lkb1 and Threonine to Glutamic Acid mutation at residue 196 of salt inducible kinase 3 in Drosophila viability have been observed. Sleep was reduced in mice after virally mediated reduction of Lkb1 in the brain. Electroencephalography analysis showed that nonrapid eye movement sleep and sleep need were both reduced in Lkb1-mutant mice. These results indicate that liver kinase B1 plays a physiological role in sleep regulation conserved from flies to mice. [ABSTRACT FROM AUTHOR]

Mutations in PARK6 (PINK1) and PARK2 (Parkin) are linked to rare familial cases of Parkinson's disease (PD). Mutations in these genes result in pathological dysregulation of mitophagy, contributing to neurodegeneration. Here, we report that environmental factors causing a specific posttranslational modification on PINK1 can mimic these genetic mutations. We describe a molecular mechanism for impairment of mitophagy via formation of S-nitrosylated PINK1 (SNO-PINK1). Mitochondrial insults simulating age- or environmental-related stress lead to increased SNO-PINK1, inhibiting its kinase activity. SNO-PINK1 decreases Parkin translocation to mitochondrial membranes, disrupting mitophagy in cell lines and human-iPSC-derived neurons. We find levels of SNO-PINK1 in brains of α-synuclein transgenic PD mice similar to those in cell-based models, indicating the pathophysiological relevance of our findings. Importantly, SNO-PINK1-mediated deficits in mitophagy contribute to neuronal cell death. These results reveal a direct molecular link between nitrosative stress, SNO-PINK1 formation, and mitophagic dysfunction that contributes to the pathogenesis of PD.

The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.

Mutations in the mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) cause Parkinsons disease (PD), likely by disrupting PINK1s kinase activity. Although the mechanism(s) underlying how this loss of activity causes degeneration remains unclear, increasing PINK1 activity may therapeutically benefit some forms of PD. However, we must first learn whether restoring PINK1 function prevents degeneration in patients harboring PINK1 mutations, or whether boosting PINK1 function can offer protection in more common causes of PD. To test these hypotheses in preclinical rodent models of PD, we used kinetin triphosphate, a small-molecule that activates both wild-type and mutant forms of PINK1, which affects mitochondrial function and protects neural cells in culture. We chronically fed kinetin, the precursor of kinetin triphosphate, to PINK1-null rats in which PINK1 was reintroduced into their midbrain, and also to rodent models overexpressing α-synuclein. The highest tolerated dose of oral kinetin increased brain levels of kinetin for up to 6 months, without adversely affecting the survival of nigrostriatal dopamine neurons. However, there was no degeneration of midbrain dopamine neurons lacking PINK1, which precluded an assessment of neuroprotection and raised questions about the robustness of the PINK1 KO rat model of PD. In two rodent models of α-synuclein-induced toxicity, boosting PINK1 activity with oral kinetin provided no protective effects. Our results suggest that oral kinetin is unlikely to protect against α-synuclein toxicity, and thus fail to provide evidence that kinetin will protect in sporadic models of PD. Kinetin may protect in cases of PINK1 deficiency, but this possibility requires a more robust PINK1 KO model that can be validated by proof-of-principle genetic correction in adult animals.

Mutations in the mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) cause Parkinson's disease (PD), likely by disrupting PINK1's kinase activity. Although the mechanism(s) underlying how this loss of activity causes degeneration remains unclear, increasing PINK1 activity may therapeutically benefit some forms of PD. However, we must first learn whether restoring PINK1 function prevents degeneration in patients harboring PINK1 mutations, or whether boosting PINK1 function can offer protection in more common causes of PD. To test these hypotheses in preclinical rodent models of PD, we used kinetin triphosphate, a small-molecule that activates both wild-type and mutant forms of PINK1, which affects mitochondrial function and protects neural cells in culture. We chronically fed kinetin, the precursor of kinetin triphosphate, to PINK1-null rats in which PINK1 was reintroduced into their midbrain, and also to rodent models overexpressing α-synuclein. The highest tolerated dose of oral kinetin increased brain levels of kinetin for up to 6 months, without adversely affecting the survival of nigrostriatal dopamine neurons. However, there was no degeneration of midbrain dopamine neurons lacking PINK1, which precluded an assessment of neuroprotection and raised questions about the robustness of the PINK1 KO rat model of PD. In two rodent models of α-synuclein-induced toxicity, boosting PINK1 activity with oral kinetin provided no protective effects. Our results suggest that oral kinetin is unlikely to protect against α-synuclein toxicity, and thus fail to provide evidence that kinetin will protect in sporadic models of PD. Kinetin may protect in cases of PINK1 deficiency, but this possibility requires a more robust PINK1 KO model that can be validated by proof-of-principle genetic correction in adult animals.

Background: Oxidative stress has a significant role in the commencement and development of hyperglycemia. Vanadium, as a transitional metal with redox properties, enters the redox process, produces free radicals, and distracts the pro-antioxidant balance. The present animal systematic review aimed to assess the effect of vanadium supplementation on inflammation and oxidative stress biomarkers in diabetes-induced animals. Methods: A systematic search was conducted using the PubMed, Scopus, and web of science databases from 1990 to 2021, according to the inclusion and exclusion criteria. The search strategy was based on the guidelines for systematic review of animal experiments and Preferred Reporting Items for Systematic Reviews (PRISMA). Criteria for eligibility were animal-based studies, evaluating the therapeutic effects of vanadium on inflammatory and oxidative stress biomarkers in diabetes. The Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool was used for assessing the methodological quality of included studies. Results: In the present study, 341 articles were evaluated out of which 42 studies were eligible for inclusion. The majority of the studies confirmed the advantageous properties of vanadium on inflammatory and oxidative stress biomarkers. A minor risk of bias was reported, based on the SYRCLE's tool. Conclusion: According to the findings, well-designed clinical trials are warranted to assess the long-lasting effects of various vanadium compounds on inflammatory and oxidative stress biomarkers. [ABSTRACT FROM AUTHOR]

Alterations in gene dosage due to copy number variation are associated with autism spectrum disorder, intellectual disability (ID), and other psychiatric disorders. The nervous system is so acutely sensitive to the dose of methyl-CpG-binding protein 2 (MeCP2) that even a twofold change in MeCP2 protein-either increased or decreased-results in distinct disorders with overlapping features including ID, autistic behavior, and severe motor dysfunction. Rett syndrome is caused by loss-of-function mutations in MECP2, whereas duplications spanning the MECP2 locus result in MECP2 duplication syndrome (MDS), which accounts for ~1% of X-linked ID. Despite evidence from mouse models that restoring MeCP2 can reverse the course of disease, there are currently no U.S. Food and Drug Administration-approved therapies available to clinically modulate MeCP2 abundance. We used a forward genetic screen against all known human kinases and phosphatases to identify druggable regulators of MeCP2 stability. Two putative modulators of MeCP2, HIPK2 (homeodomain-interacting protein kinase 2) and PP2A (protein phosphatase 2A), were validated as stabilizers of MeCP2 in vivo. Further, pharmacological inhibition of PP2A in vivo reduced MeCP2 in the nervous system and rescued both overexpression and motor abnormalities in a mouse model of MDS. Our findings reveal potential therapeutic targets for treating disorders of altered MECP2 dosage.

In this issue of Cell Chemical Biology, Zhang et al. (2017) report a zebrafish model of Parkinson's disease (PD), incorporating the PD-protein PINK1 and rotenone, a toxin linked to PD. Using it as a drug-screening platform, they identify trifluoperazine and other piperazine phenothiazines as protective compounds that enhance autophagy independent of PINK1.

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate.

The expertise of protein kinases lies in their dynamic structure, wherein they are able to modulate cellular signaling by their phosphotransferase activity. Only a few hundreds of protein kinases regulate key processes in human cells, and protein kinases play a pivotal role in health and disease. The present study dwells on understanding the working of the protein kinase-molecular switch as an allosteric network of "communities" composed of congruently dynamic residues that make up the protein kinase core. Girvan-Newman algorithm-based community maps of the kinase domain of cAMP-dependent protein kinase A allow for a molecular explanation for the role of protein conformational entropy in its catalytic cycle. The community map of a mutant, Y204A, is analyzed vis-à-vis the wild-type protein to study the perturbations in its dynamic profile such that it interferes with transfer of the γ-phosphate to a protein substrate. Conventional biochemical measurements are used to ascertain the effect of these dynamic perturbations on the kinetic profiles of both proteins. These studies pave the way for understanding how mutations far from the kinase active site can alter its dynamic properties and catalytic function even when major structural perturbations are not obvious from static crystal structures.

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIβ or the RIIβ subunit results in distinct neuronal phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co-localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIβ in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIβ, but not of RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.

Enhanced coverage and sensitivity of next-generation 'omic' platforms has allowed the characterization of gene, metabolite and protein responses in highly metabolic tissues, such as, skeletal muscle. A limitation, however, is the capability to determine interaction between dynamic biological networks. To address this limitation, we applied Weighted Analyte Correlation Network Analysis (WACNA) to RNA-seq and metabolomic datasets to identify correlated subnetworks of transcripts and metabolites in response to a high-fat diet (HFD)-induced obesity and/or exercise. HFD altered skeletal muscle lipid profiles and up-regulated genes involved in lipid catabolism, while decreasing 241 exercise-responsive genes related to skeletal muscle plasticity. WACNA identified the interplay between transcript and metabolite subnetworks linked to lipid metabolism, inflammation and glycerophospholipid metabolism that were associated with IL6, AMPK and PPAR signal pathways. Collectively, this novel experimental approach provides an integrative resource to study transcriptional and metabolic networks in skeletal muscle in the context of health and disease.

Plant-parasitic nematodes are destructive pests causing losses of billions of dollars annually. An effective plant defence against pathogens relies on the recognition of pathogen-associated molecular patterns (PAMPs) by surface-localised receptors leading to the activation of PAMP-triggered immunity (PTI). Extensive studies have been conducted to characterise the role of PTI in various models of plant-pathogen interactions. However, far less is known about the role of PTI in roots in general and in plant-nematode interactions in particular. Here we show that nematode-derived proteinaceous elicitor/s is/are capable of inducing PTI in Arabidopsis in a manner dependent on the common immune co-receptor BAK1. Consistent with the role played by BAK1, we identified a leucine-rich repeat receptor-like kinase, termed NILR1 that is specifically regulated upon infection by nematodes. We show that NILR1 is essential for PTI responses initiated by nematodes and nilr1 loss-of-function mutants are hypersusceptible to a broad category of nematodes. To our knowledge, NILR1 is the first example of an immune receptor that is involved in induction of basal immunity (PTI) in plants or in animals in response to nematodes. Manipulation of NILR1 will provide new options for nematode control in crop plants in future.

The very existence of extracellular phosphorylation has been questioned for a long time, although casein phosphorylation was discovered a century ago. In addition, several modification sites localized on secreted proteins or on extracellular or lumenal domains of transmembrane proteins have been catalogued in large scale phosphorylation analyses, though in most such studies this aspect of cellular localization was not considered. Our review presents examples when additional analyses were performed on already public data sets that revealed a wealth of information about this "neglected side" of the modification. We also sum up accumulated knowledge about extracellular phosphorylation, including the discovery of Golgi-residing kinases and the special difficulties encountered in targeted analyses. We hope future phosphorylation studies will not ignore the existence of phosphorylation outside of the cell, and further discoveries will shed more light on its biological role.

BackgroundTribbles proteins are conserved pseudokinases that function to control kinase signalling and transcription in diverse biological processes. Abnormal function in human Tribbles has been implicated in a number of diseases including leukaemia, metabolic syndromes and cardiovascular diseases. Caenorhabditis elegans Tribbles NIPI-3 was previously shown to activate host defense upon infection by promoting the conserved PMK-1/p38 mitogen-activated protein kinase (MAPK) signalling pathway. Despite the prominent role of Tribbles proteins in many species, our knowledge of their mechanism of action is fragmented, and the in vivo functional relevance of their interactions with other proteins remains largely unknown.ResultsHere, by characterizing nipi-3 null mutants, we show that nipi-3 is essential for larval development and viability. Through analyses of genetic suppressors of nipi-3 null mutant lethality, we show that NIPI-3 negatively controls PMK-1/p38 signalling via transcriptional repression of the C/EBP transcription factor CEBP-1. We identified CEBP-1's transcriptional targets by ChIP-seq analyses and found them to be enriched in genes involved in development and stress responses. Unlike its cell-autonomous role in innate immunity, NIPI-3 is required in multiple tissues to control organismal development.ConclusionsTogether, our data uncover an unprecedented crosstalk involving multiple tissues, in which NIPI-3 acts as a master regulator to inhibit CEBP-1 and the PMK-1/p38 MAPK pathway. In doing so, it keeps innate immunity in check and ensures proper organismal development.

The 'pseudokinase' SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure of Danio rerio POMK in complex with Mg2+ ions, ADP, aluminum fluoride, and the GalNAc-β3-GlcNAc-β4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-β3-GlcNAc-β4-Man is recognized by a surface groove, and the GalNAc-β3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants in POMK knockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome.

Asymmetric divisions produce daughter cells with different fates, and thus are critical for animal development. During asymmetric divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types, a cortically localized complex consisting of Gα, GPR-1/2, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynactin/dynein, which exerts pulling forces on astral microtubules to physically position the spindle. The conserved PAR polarity proteins are known to regulate both cytoplasmic asymmetry and spindle positioning in many cases. However, spindle positioning also occurs in response to cell signaling cues that appear to be PAR-independent. In the four-cell Caenorhabditis elegans embryo, Wnt and Mes-1/Src-1 signaling pathways act partially redundantly to align the spindle on the anterior/posterior axis of the endomesodermal (EMS) precursor cell. It is unclear how those extrinsic signals individually contribute to spindle positioning and whether either pathway acts via conserved spindle positioning regulators. Here, we genetically test the involvement of Gα, LIN-5, and their negative regulator LET-99, in transducing EMS spindle positioning polarity cues. We also examined whether the C. elegans ortholog of another spindle positioning regulator, DLG-1, is required. We show that LET-99 acts in the Mes-1/Src-1 pathway for spindle positioning. LIN-5 is also required for EMS spindle positioning, possibly through a Gα- and DLG-1-independent mechanism.