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There is an ongoing need for antifungal agents to treat humans. Identification of new antifungal agents can be based on screening compounds using whole cell assays. Screening compounds that target a particular molecule is possible in budding yeast wherein sophisticated strain engineering allows for controlled expression of endogenous or heterologous genes. We have considered the yeast Mps1 protein kinase as a reasonable target for antifungal agents because mutant or druggable forms of the protein, upon inactivation, cause rapid loss of cell viability. Furthermore, extensive analysis of the Mps1 in budding yeast has offered potential tactics for identifying inhibitors of its enzymatic activity. One such tactic is based on the finding that overexpression of Mps1 leads to cell cycle arrest via activation of the spindle assembly checkpoint. We have endeavored to adapt this assay to be based on the overexpression of Mps1 orthologs from pathogenic yeast in hopes of having a whole-cell assay system to test the activity of these orthologs. Mps1 orthologous genes from seven pathogenic yeast or other pathogenic fungal species were isolated and expressed in budding yeast. Two orthologs clearly produced phenotypes similar to those produced by the overexpression of budding yeast Mps1, indicating that this system for heterologous Mps1 expression has potential as a platform for identifying prospective antifungal agents.

Under low-potassium (K+) stress, a Ca2+ signaling network consisting of calcineurin B-like proteins (CBLs) and CBL-interacting kinases (CIPKs) play essential roles. Specifically, the plasma membrane CBL1/9-CIPK pathway and the tonoplast CBL2/3-CIPK pathway promotes K+ uptake and remobilization, respectively, by activating a series of K+ channels. While the dual CBL-CIPK pathways enable plants to cope with low-K+ stress, little is known about the early events that link external K+ levels to the CBL-CIPK proteins. Here we show that K+ status regulates the protein abundance and phosphorylation of the CBL-CIPK-channel modules. Further analysis revealed low K+-induced activation of VM-CBL2/3 happened earlier and was required for full activation of PM-CBL1/9 pathway. Moreover, we identified CIPK9/23 kinases to be responsible for phosphorylation of CBL1/9/2/3 in plant response to low-K+ stress and the HAB1/ABI1/ABI2/PP2CA phosphatases to be responsible for CBL2/3-CIPK9 dephosphorylation upon K+-repletion. Further genetic analysis showed that HAB1/ABI1/ABI2/PP2CA phosphatases are negative regulators for plant growth under low-K+, countering the CBL-CIPK network in plant response and adaptation to low-K+ stress.

Cyclin-dependent kinases (CDKs) are key mediators of cell proliferation and have been a subject of oncology drug discovery efforts for over two decades. Several CDK and activator cyclin family members have been implicated in regulating the cell division cycle. While it is thought that there are canonical CDK-cyclin pairing preferences, the extent of selectivity is unclear, and increasing evidence suggests that the cell-cycle CDKs can be activated by a pool of available cyclins. The molecular details of CDK-cyclin specificity are not completely understood despite their importance for understanding cancer cell cycles and for pharmacological inhibition of cancer proliferation. We report here a biolayer interferometry assay that allows for facile quantification of CDK binding interactions with their cyclin activators. We applied this assay to measure the impact of Cdk2 inhibitors on Cyclin A (CycA) association and dissociation kinetics. We found that Type I inhibitors increase the affinity between Cdk2 and CycA by virtue of a slowed cyclin dissociation rate. In contrast, Type II inhibitors and other small-molecule Cdk2 binders have distinct effects on the CycA association and dissociation processes to decrease affinity. We propose that the differential impact of small molecules on the cyclin binding kinetics arises from the plasticity of the Cdk2 active site as the kinase transitions between active, intermediate, and inactive states.

While several computational methods have been developed to predict the functional relevance of phosphorylation sites, experimental analysis of the interdependency between protein phosphorylation and Protein-Protein Interactions (PPIs) remains challenging. Here, we describe an experimental strategy to establish interdependencies between protein phosphorylation and complex formation. This strategy is based on three main steps: (i) systematically charting the phosphorylation landscape of a target protein; (ii) assigning distinct proteoforms of the target protein to different protein complexes by native complex separation (AP-BNPAGE) and protein correlation profiling; and (iii) analyzing proteoforms and complexes in cells lacking regulators of the target protein. We applied this strategy to YAP1, a transcriptional co-activator for the control of organ size and tissue homeostasis that is highly phosphorylated and among the most connected proteins in human cells. We identified multiple YAP1 phosphosites associated with distinct complexes and inferred how both are controlled by Hippo pathway members. We detected a PTPN14/LATS1/YAP1 complex and suggest a model how PTPN14 inhibits YAP1 via augmenting WW domain-dependent complex formation and phosphorylation by LATS1/2.

Leucine Rich Repeat Kinase 1 and 2 (LRRK1 and LRRK2) are homologs in the ROCO family of proteins in humans. Despite their shared domain architecture and involvement in intracellular trafficking, their disease associations are strikingly different: LRRK2 is involved in familial Parkinsons disease while LRRK1 is linked to bone diseases. Furthermore, Parkinsons disease-linked mutations in LRRK2 are typically autosomal dominant gain-of-function while those in LRRK1 are autosomal recessive loss-of-function. Here, to understand these differences, we solved cryo-EM structures of LRRK1 in its monomeric and dimeric forms. Both differ from the corresponding LRRK2 structures. Unlike LRRK2, which is sterically autoinhibited as a monomer, LRRK1 is sterically autoinhibited in a dimer-dependent manner. LRRK1 has an additional level of autoinhibition that prevents activation of the kinase and is absent in LRRK2. Finally, we place the structural signatures of LRRK1 and LRRK2 in the context of the evolution of the LRRK family of proteins.

Yes-associated protein (YAP) and its homologue TAZ are transducers of several biochemical and biomechanical signals, integrating multiplexed inputs from the microenvironment into higher level cellular functions such as proliferation, differentiation and migration. Emerging evidence suggests that Ca2+ is a key second messenger that connects microenvironmental input signals and YAP/TAZ regulation. However, studies that directly modulate Ca2+ have reported contradictory YAP/TAZ responses: in some studies, a reduction in Ca2+ influx increases the activity of YAP/TAZ, while in others, an increase in Ca2+ influx activates YAP/TAZ. Importantly, Ca2+ and YAP/TAZ exhibit distinct spatiotemporal dynamics, making it difficult to unravel their connections from a purely experimental approach. In this study, we developed a network model of Ca2+ -mediated YAP/TAZ signalling to investigate how temporal dynamics and crosstalk of signalling pathways interacting with Ca2+ can alter the YAP/TAZ response, as observed in experiments. By including six signalling modules (e.g. GPCR, IP3-Ca2+ , kinases, RhoA, F-actin and Hippo-YAP/TAZ) that interact with Ca2+ , we investigated both transient and steady-state cell response to angiotensin II and thapsigargin stimuli. The model predicts that stimuli, Ca2+ transients and frequency-dependent relationships between Ca2+ and YAP/TAZ are primarily mediated by cPKC, DAG, CaMKII and F-actin. Simulation results illustrate the role of Ca2+ dynamics and CaMKII bistable response in switching the direction of changes in Ca2+ -induced YAP/TAZ activity. A frequency-dependent YAP/TAZ response revealed the competition between upstream regulators of LATS1/2, leading to the YAP/TAZ non-monotonic response to periodic GPCR stimulation. This study provides new insights into underlying mechanisms responsible for the controversial Ca2+ -YAP/TAZ relationship observed in experiments. KEY POINTS: YAP/TAZ integrates biochemical and biomechanical inputs to regulate cellul

Protein kinases have proven to be a very productive class of therapeutic targets, and over 90 inhibitors are currently in clinical use primarily for the treatment of cancer. Repurposing these inhibitors as antimalarials could provide an accelerated path to drug development. In this study, we identified BI-2536, a known potent human polo-like kinase 1 inhibitor, with low nanomolar antiplasmodial activity. Screening of additional PLK1 inhibitors revealed further antiplasmodial candidates despite the lack of an obvious orthologue of PLKs in Plasmodium. A subset of these inhibitors was profiled for their in vitro killing profile, and commonalities between the killing rate and inhibition of nuclear replication were noted. A kinase panel screen identified PfNEK3 as a shared target of these PLK1 inhibitors; however, phosphoproteome analysis confirmed distinct signaling pathways were disrupted by two structurally distinct inhibitors, suggesting PfNEK3 may not be the sole target. Genomic analysis of BI-2536-resistant parasites revealed mutations in genes associated with the starvation-induced stress response, suggesting BI-2536 may also inhibit an aminoacyl-tRNA synthetase.

In this article, we describe the development of the plant immunity field, starting with efforts to understand the genetic basis for disease resistance, which ∼30 y ago led to the discovery of diverse classes of immune receptors that recognize and respond to infectious microbes. We focus on knowledge gained from studies of the rice XA21 immune receptor that recognizes RaxX (required for activation of XA21 mediated immunity X), a sulfated microbial peptide secreted by the gram-negative bacterium Xanthomonas oryzae pv. oryzae. XA21 is representative of a large class of plant and animal immune receptors that recognize and respond to conserved microbial molecules. We highlight the complexity of this large class of receptors in plants, discuss a possible role for RaxX in Xanthomonas biology, and draw attention to the important role of sulfotyrosine in mediating receptor-ligand interactions.

Aims/hypothesisDiabetic kidney disease (DKD) is the leading cause of kidney failure and has a substantial genetic component. Our aim was to identify novel genetic factors and genes contributing to DKD by performing meta-analysis of previous genome-wide association studies (GWAS) on DKD and by integrating the results with renal transcriptomics datasets.MethodsWe performed GWAS meta-analyses using ten phenotypic definitions of DKD, including nearly 27,000 individuals with diabetes. Meta-analysis results were integrated with estimated quantitative trait locus data from human glomerular (N=119) and tubular (N=121) samples to perform transcriptome-wide association study. We also performed gene aggregate tests to jointly test all available common genetic markers within a gene, and combined the results with various kidney omics datasets.ResultsThe meta-analysis identified a novel intronic variant (rs72831309) in the TENM2 gene associated with a lower risk of the combined chronic kidney disease (eGFR<60 ml/min per 1.73 m2) and DKD (microalbuminuria or worse) phenotype (p=9.8×10-9; although not withstanding correction for multiple testing, p>9.3×10-9). Gene-level analysis identified ten genes associated with DKD (COL20A1, DCLK1, EIF4E, PTPRN-RESP18, GPR158, INIP-SNX30, LSM14A and MFF; p<2.7×10-6). Integration of GWAS with human glomerular and tubular expression data demonstrated higher tubular AKIRIN2 gene expression in individuals with vs without DKD (p=1.1×10-6). The lead SNPs within six loci significantly altered DNA methylation of a nearby CpG site in kidneys (p<1.5×10-11). Expression of lead genes in kidney tubules or glomeruli correlated with relevant pathological phenotypes (e.g. TENM2 expression correlated positively with eGFR [p=1.6×10-8] and negatively with tubulointerstitial fibrosis [p=2.0×10-9], tubular DCLK1 expression correlated positively with fibrosis [p=7.4×10-16], and SNX30 expression correlated positively with eGFR [p=5.8×10-14] and negatively

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

Here, we show that alchemical free energy calculations can quantitatively compute the effect of mutations at the protein-protein interface. As a test case, we have used the protein complex formed by the small Rho-GTPase CDC42 and its downstream effector PAK1, a serine/threonine kinase. Notably, the CDC42/PAK1 complex offers a wealth of structural, mutagenesis, and binding affinity data because of its central role in cellular signaling and cancer progression. In this context, we have considered 16 mutations in the CDC42/PAK1 complex and obtained excellent agreement between computed and experimental data on binding affinity. Importantly, we also show that a careful analysis of the side-chain conformations in the mutated amino acids can considerably improve the computed estimates, solving issues related to sampling limitations. Overall, this study demonstrates that alchemical free energy calculations can conveniently be integrated into the design of experimental mutagenesis studies.

Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1-10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11-16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17-20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

Aims/hypothesisDiabetic kidney disease (DKD) is the leading cause of kidney failure and has a substantial genetic component. Our aim was to identify novel genetic factors and genes contributing to DKD by performing meta-analysis of previous genome-wide association studies (GWAS) on DKD and by integrating the results with renal transcriptomics datasets.MethodsWe performed GWAS meta-analyses using ten phenotypic definitions of DKD, including nearly 27,000 individuals with diabetes. Meta-analysis results were integrated with estimated quantitative trait locus data from human glomerular (N=119) and tubular (N=121) samples to perform transcriptome-wide association study. We also performed gene aggregate tests to jointly test all available common genetic markers within a gene, and combined the results with various kidney omics datasets.ResultsThe meta-analysis identified a novel intronic variant (rs72831309) in the TENM2 gene associated with a lower risk of the combined chronic kidney disease (eGFR<60 ml/min per 1.73 m2) and DKD (microalbuminuria or worse) phenotype (p=9.8×10-9; although not withstanding correction for multiple testing, p>9.3×10-9). Gene-level analysis identified ten genes associated with DKD (COL20A1, DCLK1, EIF4E, PTPRN-RESP18, GPR158, INIP-SNX30, LSM14A and MFF; p<2.7×10-6). Integration of GWAS with human glomerular and tubular expression data demonstrated higher tubular AKIRIN2 gene expression in individuals with vs without DKD (p=1.1×10-6). The lead SNPs within six loci significantly altered DNA methylation of a nearby CpG site in kidneys (p<1.5×10-11). Expression of lead genes in kidney tubules or glomeruli correlated with relevant pathological phenotypes (e.g. TENM2 expression correlated positively with eGFR [p=1.6×10-8] and negatively with tubulointerstitial fibrosis [p=2.0×10-9], tubular DCLK1 expression correlated positively with fibrosis [p=7.4×10-16], and SNX30 expression correlated positively with eGFR [p=5.8×10-14] and negatively

Here, we show that alchemical free energy calculations can quantitatively compute the effect of mutations at the protein-protein interface. As a test case, we have used the protein complex formed by the small Rho-GTPase CDC42 and its downstream effector PAK1, a serine/threonine kinase. Notably, the CDC42/PAK1 complex offers a wealth of structural, mutagenesis, and binding affinity data because of its central role in cellular signaling and cancer progression. In this context, we have considered 16 mutations in the CDC42/PAK1 complex and obtained excellent agreement between computed and experimental data on binding affinity. Importantly, we also show that a careful analysis of the side-chain conformations in the mutated amino acids can considerably improve the computed estimates, solving issues related to sampling limitations. Overall, this study demonstrates that alchemical free energy calculations can conveniently be integrated into the design of experimental mutagenesis studies.

BackgroundThe role of bacterial symbionts that populate octocorals (Cnidaria, Octocorallia) is still poorly understood. To shed light on their metabolic capacities, we examined 66 high-quality metagenome-assembled genomes (MAGs) spanning 30 prokaryotic species, retrieved from microbial metagenomes of three octocoral species and seawater.ResultsSymbionts of healthy octocorals were affiliated with the taxa Endozoicomonadaceae, Candidatus Thioglobaceae, Metamycoplasmataceae, unclassified Pseudomonadales, Rhodobacteraceae, unclassified Alphaproteobacteria and Ca. Rhabdochlamydiaceae. Phylogenomics inference revealed that the Endozoicomonadaceae symbionts uncovered here represent two species of a novel genus unique to temperate octocorals, here denoted Ca. Gorgonimonas eunicellae and Ca. Gorgonimonas leptogorgiae. Their genomes revealed metabolic capacities to thrive under suboxic conditions and high gene copy numbers of serine-threonine protein kinases, type 3-secretion system, type-4 pili, and ankyrin-repeat proteins, suggesting excellent capabilities to colonize, aggregate, and persist inside their host. Contrarily, MAGs obtained from seawater frequently lacked symbiosis-related genes. All Endozoicomonadaceae symbionts harbored endo-chitinase and chitin-binging protein-encoding genes, indicating that they can hydrolyze the most abundant polysaccharide in the oceans. Other symbionts, including Metamycoplasmataceae and Ca. Thioglobaceae, may assimilate the smaller chitin oligosaccharides resulting from chitin breakdown and engage in chitin deacetylation, respectively, suggesting possibilities for substrate cross-feeding and a role for the coral microbiome in overall chitin turnover. We also observed sharp differences in secondary metabolite production potential between symbiotic lineages. Specific Proteobacteria taxa may specialize in chemical defense and guard other symbionts, including Endozoicomonadaceae, which lack such capacity.ConclusionThis is the first study to

Many neurodevelopmental disorders (NDDs) are the result of mutations on the X chromosome. One severe NDD resulting from mutations on the X chromosome is CDKL5 deficiency disorder (CDD). CDD is an epigenetic, X-linked NDD characterized by intellectual disability (ID), pervasive seizures and severe sleep disruption, including recurring hospitalizations. CDD occurs at a 4:1 ratio, with a female bias. CDD is driven by the loss of cyclin-dependent kinase-like 5 (CDKL5), a serine/threonine kinase that is essential for typical brain development, synapse formation and signal transmission. Previous studies focused on male subjects from animal models, likely to avoid the complexity of X mosaicism. For the first time, we report translationally relevant behavioral phenotypes in young adult (8-20 weeks) females and males with robust signal size, including impairments in learning and memory, substantial hyperactivity and increased susceptibility to seizures/reduced seizure thresholds, in both sexes, and in two models of CDD preclinical mice, one with a general loss-of-function mutation and one that is a patient-derived mutation.

In this article, we describe the development of the plant immunity field, starting with efforts to understand the genetic basis for disease resistance, which ∼30 y ago led to the discovery of diverse classes of immune receptors that recognize and respond to infectious microbes. We focus on knowledge gained from studies of the rice XA21 immune receptor that recognizes RaxX (required for activation of XA21 mediated immunity X), a sulfated microbial peptide secreted by the gram-negative bacterium Xanthomonas oryzae pv. oryzae. XA21 is representative of a large class of plant and animal immune receptors that recognize and respond to conserved microbial molecules. We highlight the complexity of this large class of receptors in plants, discuss a possible role for RaxX in Xanthomonas biology, and draw attention to the important role of sulfotyrosine in mediating receptor-ligand interactions.

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2911-919, a nine amino acid segment within a flexible interdomain region (LRRK2853-981), which we designate the "regulatory loop" (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2's association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2's interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase-dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a lifelong infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete, and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified trichamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase US3 in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities in both the presence and absence of a functional US3 kinase; however, fewer nucleocapsids are moving when US3 is absent, and they move for shorter periods of time before stopping, suggesting that US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12 h later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1 h postinfection (hpi) through the stimulation of a phosphatidylinositol 3-kinase (PI3K)/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. IMPORTANCE Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There is a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized respons

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.

Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.

The activation of BRASSINOSTEROID INSENSITIVE1 (BRI1) and its association with the BRI1 ASSOCIATED RECEPTOR KINASE1 (BAK1) are key steps for the initiation of the BR signaling cascade mediating hypocotyl elongation. Heat shock protein 90 (HSP90) is crucial in the regulation of signaling processes and the activation of hormonal receptors. We report that HSP90 is required for the maintenance of the BRI1 receptor at the plasma membrane (PM) and its association with the BAK1 co-receptor during BL-ligand stimulation. HSP90 mediates BR perception and signal transduction through physical interactions with BRI1 and BAK1, while chaperone depletion resulted in lower levels of BRI1 and BAK1 receptors at the PM and affected the spatial partitioning and organization of BRI1/BAK1 heterocomplexes at the PM. The BRI1/BAK1 interaction relies on the HSP90-dependent activation of the kinase domain of BRI1 which leads to the confinement of the spatial dynamics of the membrane resident BRI1 and the attenuation of the downstream signaling. This is evident by the impaired activation and transcriptional activity of BRI1 EMS SUPPRESSOR 1 (BES1) upon HSP90 depletion. Our findings provide conclusive evidence that further expands the commitment of HSP90 in BR signaling through the HSP90-mediated activation of BRI1 in the control of the BR signaling cascade in plants.

Nonalcoholic fatty liver disease (NAFLD) is a growing cause of chronic liver disease. Using a proxy NAFLD definition of chronic elevation of alanine aminotransferase (cALT) levels without other liver diseases, we performed a multiancestry genome-wide association study (GWAS) in the Million Veteran Program (MVP) including 90,408 cALT cases and 128,187 controls. Seventy-seven loci exceeded genome-wide significance, including 25 without prior NAFLD or alanine aminotransferase associations, with one additional locus identified in European American-only and two in African American-only analyses (P < 5 × 10-8). External replication in histology-defined NAFLD cohorts (7,397 cases and 56,785 controls) or radiologic imaging cohorts (n = 44,289) replicated 17 single-nucleotide polymorphisms (SNPs) (P < 6.5 × 10-4), of which 9 were new (TRIB1, PPARG, MTTP, SERPINA1, FTO, IL1RN, COBLL1, APOH and IFI30). Pleiotropy analysis showed that 61 of 77 multiancestry and all 17 replicated SNPs were jointly associated with metabolic and/or inflammatory traits, revealing a complex model of genetic architecture. Our approach integrating cALT, histology and imaging reveals new insights into genetic liability to NAFLD.

Aging is a major risk factor to develop neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors involved in synaptic function and pathways linked to neurodegenerative diseases. The genes modified by XBP1 in the aged hippocampus where also altered. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.

The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein-protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations, including the ELYS-MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.

BackgroundSinonasal Undifferentiated Carcinoma (SNUC) is a rare and aggressive skull base tumor with poor survival and limited treatment options. To date, targeted sequencing studies have identified IDH2 and SMARCB1 as potential driver alterations, but the molecular alterations found in SMARCB1 wild type tumors are unknown.MethodsWe evaluated survival outcomes in a cohort of 46 SNUC patients treated at an NCI designated cancer center and identify clinical and disease variables associated with survival on Kaplan-Meier and Cox multivariate survival analysis. We performed exome sequencing to characterize a series of SNUC tumors (n = 5) and cell line (MDA8788-6) to identify high confidence mutations, copy number alterations, microsatellite instability, and fusions. Knockdown studies using siRNA were utilized for validation of a novel PGAP3-SRPK1 gene fusion.ResultsOverall survival analysis revealed no significant difference in outcomes between patients treated with surgery +/- CRT and CRT alone. Tobacco use was the only significant predictor of survival. We also confirmed previously published findings on IDH and SMARC family mutations and identified novel recurrent aberrations in the JAK/STAT and PI3K pathways. We also validated a novel PGAP3-SRPK1 gene fusion in the SNUC cell line, and show that knockdown of the fusion is negatively associated with EGFR, E2F and MYC signaling.ConclusionCollectively, these data demonstrate recurrent alterations in the SWI/SNF family as well as IDH, JAK/STAT, and PI3K pathways and discover a novel fusion gene (PGAP3-SRPK1). These data aim to improve understanding of possible driver mutations and guide future therapeutic strategies for this disease.

The type I interferon (IFN) pathway is a key component of innate immune response upon invasion of foreign pathogens. It is also under precise control to prevent excessive upregulation and undesired inflammation cascade. In the present study, we report that Riok3, an atypical kinase, negatively regulates retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) sensing-induced type I IFN signaling. Riok3 deficiency selectively inhibits RNA viral replication in vitro, resulting from an upregulated type I IFN pathway. Mice with myeloid-specific Riok3 knockout also show a more robust induction of type I IFN upon RNA virus infection and are more resistant to RNA virus-induced pathogenesis. Mechanistically, Riok3 recruits and interacts with the E3 ubiquitin ligase TRIM40, leading to the degradation of RIG-I and melanoma differentiation-associated gene-5 (MDA5) via K48- and K27-linked ubiquitination. Collectively, our data reveal the mechanism that Riok3 employs to be a negative regulator of antiviral innate immunity.

How tubular organs elongate is poorly understood. We found that attenuated ciliary Hedgehog signaling in the gut wall impaired patterning of the circumferential smooth muscle and inhibited proliferation and elongation of developing intestine and esophagus. Similarly, ablation of gut-wall smooth muscle cells reduced lengthening. Disruption of ciliary Hedgehog signaling or removal of smooth muscle reduced residual stress within the gut wall and decreased activity of the mechanotransductive effector YAP. Removing YAP in the mesenchyme also reduced proliferation and elongation, but without affecting smooth muscle formation, suggesting that YAP interprets the smooth muscle-generated force to promote longitudinal growth. Additionally, we developed an intestinal culture system that recapitulates the requirements for cilia and mechanical forces in elongation. Pharmacologically activating YAP in this system restored elongation of cilia-deficient intestines. Thus, our results reveal that ciliary Hedgehog signaling patterns the circumferential smooth muscle to generate radial mechanical forces that activate YAP and elongate the gut.

The type I interferon (IFN) pathway is a key component of innate immune response upon invasion of foreign pathogens. It is also under precise control to prevent excessive upregulation and undesired inflammation cascade. In the present study, we report that Riok3, an atypical kinase, negatively regulates retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) sensing-induced type I IFN signaling. Riok3 deficiency selectively inhibits RNA viral replication in vitro, resulting from an upregulated type I IFN pathway. Mice with myeloid-specific Riok3 knockout also show a more robust induction of type I IFN upon RNA virus infection and are more resistant to RNA virus-induced pathogenesis. Mechanistically, Riok3 recruits and interacts with the E3 ubiquitin ligase TRIM40, leading to the degradation of RIG-I and melanoma differentiation-associated gene-5 (MDA5) via K48- and K27-linked ubiquitination. Collectively, our data reveal the mechanism that Riok3 employs to be a negative regulator of antiviral innate immunity.

The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein-protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations, including the ELYS-MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.

BackgroundAlternative splicing is a rich source of tumor-specific neoantigen targets for immunotherapy. This holds promise for glioblastomas (GBMs), the most common primary tumors of the adult brain, which are resistant to standard-of-care therapy. Although most clinical trials enroll patients at recurrence, most preclinical studies have been done with specimens from primary disease. There are limited expression data from GBMs at recurrence and surprisingly little is known about the evolution of splicing patterns under therapy.ResultWe profile 37 primary-recurrent paired human GBM specimens via RNA sequencing. We describe the landscape of alternative splicing in GBM at recurrence and contrast that to primary and non-malignant brain-tissue specimens. By screening single-cell atlases, we identify cell-type-specific splicing patterns and novel splicing events in cell-surface proteins that are suitable targets for engineered T cell therapies. We identify recurrent-specific isoforms of mitogen-activated kinase pathway genes that enhance invasiveness and are preferentially expressed by stem-like cells.ConclusionThese studies shed light on gene expression in recurrent GBM and identify novel targets for therapeutic development.

How tubular organs elongate is poorly understood. We found that attenuated ciliary Hedgehog signaling in the gut wall impaired patterning of the circumferential smooth muscle and inhibited proliferation and elongation of developing intestine and esophagus. Similarly, ablation of gut-wall smooth muscle cells reduced lengthening. Disruption of ciliary Hedgehog signaling or removal of smooth muscle reduced residual stress within the gut wall and decreased activity of the mechanotransductive effector YAP. Removing YAP in the mesenchyme also reduced proliferation and elongation, but without affecting smooth muscle formation, suggesting that YAP interprets the smooth muscle-generated force to promote longitudinal growth. Additionally, we developed an intestinal culture system that recapitulates the requirements for cilia and mechanical forces in elongation. Pharmacologically activating YAP in this system restored elongation of cilia-deficient intestines. Thus, our results reveal that ciliary Hedgehog signaling patterns the circumferential smooth muscle to generate radial mechanical forces that activate YAP and elongate the gut.

Early-onset epileptic encephalopathies are severe disorders often associated with specific genetic mutations. In this context, the CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by early-onset seizures, intellectual delay, and motor dysfunction. Although crucial for proper brain development, the precise targets of CDKL5 and its relation to patients' symptoms are still unknown. Here, induced pluripotent stem cells derived from individuals deficient in CDKL5 protein were used to generate neural cells. Proteomic and phosphoproteomic approaches revealed disruption of several pathways, including microtubule-based processes and cytoskeleton organization. While CDD-derived neural progenitor cells have proliferation defects, neurons showed morphological alterations and compromised glutamatergic synaptogenesis. Moreover, the electrical activity of CDD cortical neurons revealed hyperexcitability during development, leading to an overly synchronized network. Many parameters of this hyperactive network were rescued by lead compounds selected from a human high-throughput drug screening platform. Our results enlighten cellular, molecular, and neural network mechanisms of genetic epilepsy that could ultimately promote novel therapeutic opportunities for patients.

BackgroundThe vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses.ResultsWe studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature stop codon in ERECTA, a LRR receptor-like serine/threonine-protein kinase. Mutations in this gene are pleiotropic indicating that it serves multiple roles during plant development. This mutant also displayed changes in cell wall composition, gene expression and hormone homeostasis.ConclusionIn summary, ERECTA has a pleiotropic role in Brachypodium. We propose a major role of ERECTA in vasculature anastomosis and vascular tissue organization in Brachypodium.

Crop damage by herbivorous insects remains a significant contributor to annual yield reductions. Following attack, maize (Zea mays) responds to herbivore-associated molecular patterns (HAMPs) and damage-associated molecular patterns (DAMPs), activating dynamic direct and indirect antiherbivore defense responses. To define underlying signaling processes, comparative analyses between plant elicitor peptide (Pep) DAMPs and fatty acid-amino acid conjugate (FAC) HAMPs were conducted. RNA sequencing analysis of early transcriptional changes following Pep and FAC treatments revealed quantitative differences in the strength of response yet a high degree of qualitative similarity, providing evidence for shared signaling pathways. In further comparisons of FAC and Pep responses across diverse maize inbred lines, we identified Mo17 as part of a small subset of lines displaying selective FAC insensitivity. Genetic mapping for FAC sensitivity using the intermated B73 × Mo17 population identified a single locus on chromosome 4 associated with FAC sensitivity. Pursuit of multiple fine-mapping approaches further narrowed the locus to 19 candidate genes. The top candidate gene identified, termed FAC SENSITIVITY ASSOCIATED (ZmFACS), encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) that belongs to the same family as a rice (Oryza sativa) receptor gene previously associated with the activation of induced responses to diverse Lepidoptera. Consistent with reduced sensitivity, ZmFACS expression was significantly lower in Mo17 as compared to B73. Transient heterologous expression of ZmFACS in Nicotiana benthamiana resulted in a significantly increased FAC-elicited response. Together, our results provide useful resources for studying early elicitor-induced antiherbivore responses in maize and approaches to discover gene candidates underlying HAMP sensitivity in grain crops.

BackgroundSinonasal Undifferentiated Carcinoma (SNUC) is a rare and aggressive skull base tumor with poor survival and limited treatment options. To date, targeted sequencing studies have identified IDH2 and SMARCB1 as potential driver alterations, but the molecular alterations found in SMARCB1 wild type tumors are unknown.MethodsWe evaluated survival outcomes in a cohort of 46 SNUC patients treated at an NCI designated cancer center and identify clinical and disease variables associated with survival on Kaplan-Meier and Cox multivariate survival analysis. We performed exome sequencing to characterize a series of SNUC tumors (n = 5) and cell line (MDA8788-6) to identify high confidence mutations, copy number alterations, microsatellite instability, and fusions. Knockdown studies using siRNA were utilized for validation of a novel PGAP3-SRPK1 gene fusion.ResultsOverall survival analysis revealed no significant difference in outcomes between patients treated with surgery +/- CRT and CRT alone. Tobacco use was the only significant predictor of survival. We also confirmed previously published findings on IDH and SMARC family mutations and identified novel recurrent aberrations in the JAK/STAT and PI3K pathways. We also validated a novel PGAP3-SRPK1 gene fusion in the SNUC cell line, and show that knockdown of the fusion is negatively associated with EGFR, E2F and MYC signaling.ConclusionCollectively, these data demonstrate recurrent alterations in the SWI/SNF family as well as IDH, JAK/STAT, and PI3K pathways and discover a novel fusion gene (PGAP3-SRPK1). These data aim to improve understanding of possible driver mutations and guide future therapeutic strategies for this disease.

In the developing embryos, the cortical polarity regulator Par-3 is critical for establishing Notch signaling asymmetry between daughter cells during asymmetric cell division (ACD). How cortically localized Par-3 establishes asymmetric Notch activity in the nucleus is not understood. Here, using in vivo time-lapse imaging of mitotic radial glia progenitors in the developing zebrafish forebrain, we uncover that during horizontal ACD along the anteroposterior embryonic axis, endosomes containing the Notch ligand DeltaD (Dld) move toward the cleavage plane and preferentially segregate into the posterior (subsequently basal) Notchhi daughter. This asymmetric segregation requires the activity of Par-3 and dynein motor complex. Using label retention expansion microscopy, we further detect Par-3 in the cytosol colocalizing the dynein light intermediate chain 1 (Dlic1) onto Dld endosomes. Par-3, Dlic1, and Dld are associated in protein complexes in vivo. Our data reveal an unanticipated mechanism by which cytoplasmic Par-3 directly polarizes Notch signaling components during ACD.

The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER). In metazoan cells, the Ire1 branch of the UPR initiates two functional outputs-non-conventional mRNA splicing and selective mRNA decay (RIDD). By contrast, Ire1 orthologs from Saccharomyces cerevisiae and Schizosaccharomyces pombe are specialized for only splicing or RIDD, respectively. Previously, we showed that the functional specialization lies in Ire1's RNase activity, which is either stringently splice-site specific or promiscuous (Li et al., 2018). Here, we developed an assay that reports on Ire1's RNase promiscuity. We found that conversion of two amino acids within the RNase domain of S. cerevisiae Ire1 to their S. pombe counterparts rendered it promiscuous. Using biochemical assays and computational modeling, we show that the mutations rewired a pair of salt bridges at Ire1 RNase domain's dimer interface, changing its protomer alignment. Thus, Ire1 protomer alignment affects its substrates specificity.

Conditional genetically engineered mouse models (GEMMs) of non-small cell lung cancer (NSCLC) harbor common oncogenic driver mutations of the disease, but in contrast to human NSCLC these models possess low tumor mutational burden (TMB). As a result, these models often lack tumor antigens that can elicit host adaptive immune responses, which limits their utility in immunotherapy studies. Here, we establish Kras-mutant murine models of NSCLC bearing the common driver mutations associated with the disease and increased TMB, by in vitro exposure of cell lines derived from GEMMs of NSCLC [KrasG12D (K), KrasG12DTp53-/-(KP), KrasG12DTp53+/-Lkb1-/- (KPL)] to the alkylating agent N-methyl-N-nitrosourea (MNU). Increasing the TMB enhanced host anti-tumor T cell responses and improved anti-PD-1 efficacy in syngeneic models across all genetic backgrounds. However, limited anti-PD-1 efficacy was observed in the KPL cell lines with increased TMB, which possessed a distinct immunosuppressed tumor microenvironment (TME) primarily composed of granulocytic myeloid-derived suppressor cells (G-MDSCs). This KPL phenotype is consistent with findings in human KRAS-mutant NSCLC where LKB1 loss is a driver of primary resistance to PD-1 blockade. In summary, these novel Kras-mutant NSCLC murine models with known driver mutations and increased TMB have distinct TMEs and recapitulate the therapeutic vulnerabilities of human NSCLC. We anticipate that these immunogenic models will facilitate the development of innovative immunotherapies in NSCLC.

CILK1 (ciliogenesis associated kinase 1)/ICK (intestinal cell kinase) is a highly conserved protein kinase that regulates primary cilia structure and function. CILK1 mutations cause a wide spectrum of human diseases collectively called ciliopathies. While several CILK1 heterozygous variants have been recently linked to juvenile myoclonic epilepsy (JME), it remains unclear whether these mutations cause seizures. Herein, we investigated whether mice harboring either a heterozygous null Cilk1 (Cilk1+/-) mutation or a heterozygous loss-of-function Cilk1 mutation (Cilk1R272Q/+) have epilepsy. We first evaluated the spontaneous seizure phenotype of Cilk1+/- and Cilk1R272Q/+ mice relative to wildtype littermates. We observed no electrographic differences among the three mouse genotypes during prolonged recordings. We also evaluated electrographic and behavioral responses of mice recovering from isoflurane anesthesia, an approach recently used to measure seizure-like activity. Again, we observed no electrographic or behavioral differences in control versus Cilk1+/- and Cilk1R272Q/+ mice upon isoflurane recovery. These results indicate that mice bearing a non-functional copy of Cilk1 fail to produce electrographic patterns resembling those of JME patients with a variant CILK1 copy. Our findings argue against CILK1 haploinsufficiency being the mechanism that links CILK1 variants to JME.

BackgroundBone loss is the most common reason for broken bones among the elderly. An ideal agent for the treatment of bone loss should have both osteoclast inhibitory and osteoblast stimulatory functions. Leucine-rich repeat kinase 1 (LRRK1) is a novel target for alternative antiresorptive drugs to treat osteoporosis and osteoporotic fractures. Recently a chemical IN04, Methyl 3-[({([5-(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-thio}-acetyl)-amino]-benzoate, has been identified as a potential LRRK1 inhibitor.ObjectiveThe aim of this work is to investigate how the chemical IN04 interacts with LRRK1 and inhibits its activity.MethodsA structural model of the LRRK1 kinase domain was constructed with SWISS-MODEL. The human protein kinase ROCO4 (PDB ID: 4YZN) was chosen as the template based on sequence homology, structural and phylogenetic analysis. In addition, a homology model of the LRRK1 ROC domain was also prepared based on the LRRK2 ROC domain structure (PDB ID: 2ZEJ). The interactions of IN04 with the active sites in the LRRK1 kinase domain and ROC domain were investigated by SwissDock.ResultsIN04 was docked into the active site of the LRRK1 kinase domain with similar interactions as ATP comparable to the ligand bound to homologous kinases. Many rational binding modes of IN04 to LRRK1 kinase domain were investigated and the most likely binding pose containing multiple hydrogen bonds and a salt bridge was discovered. However, IN04 cannot fit into the GDP-binding site of the ROC domain.ConclusionChemical IN04 inhibits LRRK1 by binding to the active site of the kinase domain but not the ROC domain.

During cell division, kinetochores couple chromosomes to spindle microtubules. To protect against chromosome gain or loss, kinetochores lacking microtubule attachment locally catalyze association of the checkpoint proteins Cdc20 and Mad2, which is the key event in the formation of a diffusible checkpoint complex that prevents mitotic exit. We elucidated the mechanism of kinetochore-catalyzed Mad2-Cdc20 assembly with a probe that specifically monitors this assembly reaction at kinetochores in living cells. We found that catalysis occurs through a tripartite mechanism that includes localized delivery of Mad2 and Cdc20 substrates and two phosphorylation-dependent interactions that geometrically constrain their positions and prime Cdc20 for interaction with Mad2. These results reveal how unattached kinetochores create a signal that ensures genome integrity during cell division.

Low-protein diets promote metabolic health in rodents and humans, and the benefits of low-protein diets are recapitulated by specifically reducing dietary levels of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we demonstrate that each BCAA has distinct metabolic effects. A low isoleucine diet reprograms liver and adipose metabolism, increasing hepatic insulin sensitivity and ketogenesis and increasing energy expenditure, activating the FGF21-UCP1 axis. Reducing valine induces similar but more modest metabolic effects, whereas these effects are absent with low leucine. Reducing isoleucine or valine rapidly restores metabolic health to diet-induced obese mice. Finally, we demonstrate that variation in dietary isoleucine levels helps explain body mass index differences in humans. Our results reveal isoleucine as a key regulator of metabolic health and the adverse metabolic response to dietary BCAAs and suggest reducing dietary isoleucine as a new approach to treating and preventing obesity and diabetes.

The exocyst is an evolutionarily conserved protein complex that regulates vesicular trafficking and scaffolds signal transduction. Key upstream components of the exocyst include monomeric RAL GTPases, which help mount cell-autonomous responses to trophic and immunogenic signals. Here, we present a quantitative proteomics-based characterization of dynamic and signal-dependent exocyst protein interactomes. Under viral infection, an Exo84 exocyst subcomplex assembles the immune kinase Protein Kinase R (PKR) together with the Hippo kinase Macrophage Stimulating 1 (MST1). PKR phosphorylates MST1 to activate Hippo signaling and inactivate Yes Associated Protein 1 (YAP1). By contrast, a Sec5 exocyst subcomplex recruits another immune kinase, TANK binding kinase 1 (TBK1), which interacted with and activated mammalian target of rapamycin (mTOR). RALB was necessary and sufficient for induction of Hippo and mTOR signaling through parallel exocyst subcomplex engagement, supporting the cellular response to virus infection and oncogenic signaling. This study highlights RALB-exocyst signaling subcomplexes as mechanisms for the integrated engagement of Hippo and mTOR signaling in cells challenged by viral pathogens or oncogenic signaling.

XA21 encodes a rice immune receptor that confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo). XA21-mediated immunity is triggered by recognition of a small protein called RaxX-sY (required for activation of XA21-mediated immunity X, tyrosine-sulfated) secreted by Xoo. To identify components regulating XA21-mediated immunity, we generated and screened a mutant population of fast-neutron-mutagenized rice expressing Ubi:Myc-XA21 for those susceptible to Xoo. Here, we report the characterization of one of these rice mutants, named sxi2 (suppressor of XA21-mediated immunity-2). Whole-genome sequencing revealed that sxi2 carries a deletion of the PALADIN (PALD) gene encoding a protein with three putative protein tyrosine phosphatase-like domains (PTP-A, -B, and -C). Expression of PALD in the sxi2 genetic background was sufficient to complement the susceptible phenotype, which requires the catalytic cysteine of the PTP-A active site to restore resistance. PALD co-immunoprecipitated with the full-length XA21 protein, whose levels are positively regulated by the presence of the PALD transgene. Furthermore, we foundd that sxi2 retains many hallmarks of XA21-mediated immunity, similar to the wild type. These results reveal that PALD, a previously uncharacterized class of phosphatase, functions in rice innate immunity, and suggest that the conserved cysteine in the PTP-A domain of PALD is required for its immune function.

Long noncoding RNAs (lncRNAs) are emerging as a new class of important regulators of signal transduction in tissue homeostasis and cancer development. Liquid-liquid phase separation (LLPS) occurs in a wide range of biological processes, while its role in signal transduction remains largely undeciphered. In this study, we uncovered a lipid-associated lncRNA, small nucleolar RNA host gene 9 (SNHG9) as a tumor-promoting lncRNA driving liquid droplet formation of Large Tumor Suppressor Kinase 1 (LATS1) and inhibiting the Hippo pathway. Mechanistically, SNHG9 and its associated phosphatidic acids (PA) interact with the C-terminal domain of LATS1, promoting LATS1 phase separation and inhibiting LATS1-mediated YAP phosphorylation. Loss of SNHG9 suppresses xenograft breast tumor growth. Clinically, expression of SNHG9 positively correlates with YAP activity and breast cancer progression. Taken together, our results uncover a novel regulatory role of a tumor-promoting lncRNA (i.e., SNHG9) in signal transduction and cancer development by facilitating the LLPS of a signaling kinase (i.e., LATS1).

Recurrent breast cancer presents significant challenges with aggressive phenotypes and treatment resistance. Therefore, novel therapeutics are urgently needed. Here, we report that murine recurrent breast tumor cells, when compared with primary tumor cells, are highly sensitive to ferroptosis. Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), the receptor for collagen I, is highly expressed in ferroptosis-sensitive recurrent tumor cells and human mesenchymal breast cancer cells. EMT regulators, TWIST and SNAIL, significantly induce DDR2 expression and sensitize ferroptosis in a DDR2-dependent manner. Erastin treatment induces DDR2 upregulation and phosphorylation, independent of collagen I. Furthermore, DDR2 knockdown in recurrent tumor cells reduces clonogenic proliferation. Importantly, both the ferroptosis protection and reduced clonogenic growth may be compatible with the compromised YAP/TAZ upon DDR2 inhibition. Collectively, these findings identify the important role of EMT-driven DDR2 upregulation in recurrent tumors in maintaining growth advantage but activating YAP/TAZ-mediated ferroptosis susceptibility, providing potential strategies to eradicate recurrent breast cancer cells with mesenchymal features.

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We found that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 has diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

BACKGROUND: Currently available structural variant (SV) detection methods do not span the complete spectrum of disease-causing SVs. Optical genome mapping (OGM), an emerging technology with the potential to resolve diagnostic dilemmas, was performed to investigate clinically-relevant SVs in a 4-year-old male with an epileptic encephalopathy of undiagnosed molecular origin. METHODS: OGM was utilized to image long, megabase-size DNA molecules, fluorescently labeled at specific sequence motifs throughout the genome with high sensitivity for detection of SVs greater than 500 bp in size. OGM results were confirmed in a CLIA-certified laboratory via mate-pair sequencing. RESULTS: OGM identified a mosaic, de novo 90 kb deletion and inversion on the X chromosome disrupting the CDKL5 gene. Detection of the mosaic deletion, which had been previously undetected by chromosomal microarray, an infantile epilepsy panel including exon-level microarray for CDKL5, exome sequencing as well as genome sequencing, resulted in a diagnosis of X-linked dominant early infantile epileptic encephalopathy-2. CONCLUSION: OGM affords an effective technology for the detection of SVs, especially those that are mosaic, since these remain difficult to detect with current NGS technologies and with conventional chromosomal microarrays. Further research in undiagnosed populations with OGM is warranted.