EVI1 drives leukemogenesis through aberrant ERG activation.

Chromosomal rearrangements involving the MDS1 and EVI1 complex locus (MECOM) on chromosome 3q26 define an aggressive subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and dismal prognosis. Established treatment regimens commonly fail in these patients, therefore, there is an urgent need for new therapeutic concepts that will require a better understanding of the molecular and cellular functions of the ecotropic viral integration site 1 (EVI1) oncogene. To characterize gene regulatory functions of EVI1 and associated dependencies in AML, we developed experimentally tractable human and murine disease models, investigated the transcriptional consequences of EVI1 withdrawal in vitro and in vivo, and performed the first genome-wide CRISPR screens in EVI1-dependent AML. By integrating conserved transcriptional targets with genetic dependency data, we identified and characterized the ETS transcription factor ERG as a direct transcriptional target of EVI1 that is aberrantly expressed and selectively required in both human and murine EVI1-driven AML. EVI1 controls the expression of ERG and occupies a conserved intragenic enhancer region in AML cell lines and samples from patients with primary AML. Suppression of ERG induces terminal differentiation of EVI1-driven AML cells, whereas ectopic expression of ERG abrogates their dependence on EVI1, indicating that the major oncogenic functions of EVI1 are mediated through aberrant transcriptional activation of ERG. Interfering with this regulatory axis may provide entry points for the development of rational targeted therapies.

Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans.

BACKGROUND: Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare. METHODS: We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast. RESULTS: We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7. CONCLUSIONS: We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).

Senescence-associated reprogramming promotes cancer stemness.

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16INK4a, p21CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eµ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.

Loss-of-Function Myeloperoxidase Mutations Are Associated with Increased Neutrophil Counts and Pustular Skin Disease.

The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031-2A>C) in MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis (c.2031-2A>C;c.2031-2A>C) and in two individuals with acute generalized exanthematous pustulosis (c.1705C>T;c.2031-2A>C and c.1552_1565del;c.1552_1565del). A subsequent analysis of UK Biobank data demonstrated that the c.2031-2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population (p = 5.1 × 10-6 and p = 3.6 × 10-5, respectively). The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles (c.995C>T [p.Ala332Val] and c.752T>C [p.Met251Thr]) yielding p values < 10-10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored.

The SCL30a SR protein regulates ABA-dependent seed traits and germination under stress.

SR proteins are conserved RNA-binding proteins best known as splicing regulators that have also been implicated in other steps of gene expression. Despite mounting evidence for a role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain poorly understood. Here we show that the plant-specific SCL30a SR protein negatively regulates ABA signaling to control seed traits and stress responses during germination in Arabidopsis. Transcriptome-wide analyses revealed that loss of SCL30a function barely affects splicing, but largely induces ABA-responsive gene expression and genes repressed during germination. Accordingly, scl30a mutant seeds display delayed germination and hypersensitivity to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA and salt stress sensitivity. An ABA biosynthesis inhibitor rescues the enhanced mutant seed stress sensitivity, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the gene promotes seed germination under stress by reducing sensitivity to the phytohormone. Our results reveal a new player in ABA-mediated control of early development and stress response.

Nonadherence to systemic immune-modifying therapy in people with psoriasis during the COVID-19 pandemic: findings from a global cross-sectional survey.

BACKGROUND: Nonadherence to immune-modifying therapy is a complex behaviour which, before the COVID-19 pandemic, was shown to be associated with mental health disorders in people with immune-mediated diseases. The COVID-19 pandemic has led to a rise in the global prevalence of anxiety and depression, and limited data exist on the association between mental health and nonadherence to immune-modifying therapy during the pandemic. OBJECTIVES: To assess the extent of and reasons underlying nonadherence to systemic immune-modifying therapy during the COVID-19 pandemic in individuals with psoriasis, and the association between mental health and nonadherence. METHODS: Online self-report surveys (PsoProtectMe), including validated screens for anxiety and depression, were completed globally during the first year of the pandemic. We assessed the association between anxiety or depression and nonadherence to systemic immune-modifying therapy using binomial logistic regression, adjusting for potential cofounders (age, sex, ethnicity, comorbidity) and country of residence. RESULTS: Of 3980 participants from 77 countries, 1611 (40.5%) were prescribed a systemic immune-modifying therapy. Of these, 408 (25.3%) reported nonadherence during the pandemic, most commonly due to concerns about their immunity. In the unadjusted model, a positive anxiety screen was associated with nonadherence to systemic immune-modifying therapy [odds ratio (OR) 1.37, 95% confidence interval (CI) 1.07-1.76]. Specifically, anxiety was associated with nonadherence to targeted therapy (OR 1.41, 95% CI 1.01-1.96) but not standard systemic therapy (OR 1.16, 95% CI 0.81-1.67). In the adjusted model, although the directions of the effects remained, anxiety was not significantly associated with nonadherence to overall systemic (OR 1.20, 95% CI 0.92-1.56) or targeted (OR 1.33, 95% CI 0.94-1.89) immune-modifying therapy. A positive depression screen was not strongly associated with nonadherence to systemic immune-modifying therapy in the unadjusted (OR 1.22, 95% CI 0.94-1.57) or adjusted models (OR 1.14, 95% CI 0.87-1.49). CONCLUSIONS: These data indicate substantial nonadherence to immune-modifying therapy in people with psoriasis during the pandemic, with attenuation of the association with mental health after adjusting for confounders. Future research in larger populations should further explore pandemic-specific drivers of treatment nonadherence. Clear communication of the reassuring findings from population-based research regarding immune-modifying therapy-associated adverse COVID-19 risks to people with psoriasis is essential, to optimize adherence and disease outcomes.

CDK6 is an essential direct target of NUP98 fusion proteins in acute myeloid leukemia.

Fusion proteins involving Nucleoporin 98 (NUP98) are recurrently found in acute myeloid leukemia (AML) and are associated with poor prognosis. Lack of mechanistic insight into NUP98-fusion-dependent oncogenic transformation has so far precluded the development of rational targeted therapies. We reasoned that different NUP98-fusion proteins deregulate a common set of transcriptional targets that might be exploitable for therapy. To decipher transcriptional programs controlled by diverse NUP98-fusion proteins, we developed mouse models for regulatable expression of NUP98/NSD1, NUP98/JARID1A, and NUP98/DDX10. By integrating chromatin occupancy profiles of NUP98-fusion proteins with transcriptome profiling upon acute fusion protein inactivation in vivo, we defined the core set of direct transcriptional targets of NUP98-fusion proteins. Among those, CDK6 was highly expressed in murine and human AML samples. Loss of CDK6 severely attenuated NUP98-fusion-driven leukemogenesis, and NUP98-fusion AML was sensitive to pharmacologic CDK6 inhibition in vitro and in vivo. These findings identify CDK6 as a conserved, critical direct target of NUP98-fusion proteins, proposing CDK4/CDK6 inhibitors as a new rational treatment option for AML patients with NUP98-fusions.

Biomarkers of disease progression in people with psoriasis: a scoping review.

BACKGROUND: Identification of those at risk of more severe psoriasis and/or associated morbidities offers opportunity for early intervention, reduced disease burden and more cost-effective healthcare. Prognostic biomarkers of disease progression have thus been the focus of intense research, but none are part of routine practice. OBJECTIVES: To identify and catalogue candidate biomarkers of disease progression in psoriasis for the translational research community. METHODS: A systematic search of CENTRAL, Embase, LILACS and MEDLINE was performed for relevant articles published between 1990 and December 2021. Eligibility criteria were studies involving patients with psoriasis (any age, n ≥ 50) reporting biomarkers associated with disease progression. The main outcomes were any measure of skin severity or any prespecified psoriasis comorbidity. Data were extracted by one reviewer and checked by a second; studies meeting minimal quality criteria (longitudinal design and/or use of methods to control for confounding) were formally assessed for bias. Candidate biomarkers were identified by an expert multistakeholder group using a majority voting consensus exercise, and mapped to relevant cellular and molecular pathways. RESULTS: Of 181 included studies, most investigated genomic or proteomic biomarkers associated with disease severity (n = 145) or psoriatic arthritis (n = 30). Methodological and reporting limitations compromised interpretation of findings, most notably a lack of longitudinal studies, and inadequate control for key prognostic factors. The following candidate biomarkers with future potential utility were identified for predicting disease severity: LCE3D, interleukin (IL)23R, IL23A, NFKBIL1 loci, HLA-C*06:02 (genomic), IL-17A, IgG aHDL, GlycA, I-FABP and kallikrein 8 (proteomic), tyramine (metabolomic); psoriatic arthritis: HLA-C*06:02, HLA-B*27, HLA-B*38, HLA-B*08, and variation at the IL23R and IL13 loci (genomic); IL-17A, CXCL10, Mac-2 binding protein, integrin b5, matrix metalloproteinase-3 and macrophage-colony stimulating factor (proteomic) and tyramine and mucic acid (metabolomic); and type 2 diabetes mellitus: variation in IL12B and IL23R loci (genomic). No biomarkers were supported by sufficient evidence for clinical use without further validation. CONCLUSIONS: This review provides a comprehensive catalogue of investigated biomarkers of disease progression in psoriasis. Future studies must address the common methodological limitations identified herein to expedite discovery and validation of biomarkers for clinical use. What is already known about this topic? The current treatment paradigm in psoriasis is reactive. There is a need to develop effective risk-stratified management approaches that can proactively attenuate the substantial burden of disease. Prognostic biomarkers of disease progression have therefore been the focus of intense research. What does this study add? This review is the first to scope, collate and catalogue research investigating biomarkers of disease progression in psoriasis. The review identifies potentially promising candidate biomarkers for further investigation and highlights common important limitations that should be considered when designing and conducting future studies in this area.

Biomarkers of systemic treatment response in people with psoriasis: a scoping review.

BACKGROUND: Responses to the systemic treatments commonly used to treat psoriasis vary. Biomarkers that accurately predict effectiveness and safety would enable targeted treatment selection, improved patient outcomes and more cost-effective healthcare. OBJECTIVES: To perform a scoping review to identify and catalogue candidate biomarkers of systemic treatment response in psoriasis for the translational research community. METHODS: A systematic search of CENTRAL, Embase, LILACS and MEDLINE was performed for relevant articles published between 1990 and December 2021. Eligibility criteria were studies involving patients with psoriasis (any age, n ≥ 50) reporting biomarkers associated with systemic treatment response. The main outcomes were any measure of systemic treatment efficacy or safety. Data were extracted by one reviewer and checked by a second; studies meeting minimal quality criteria (use of methods to control for confounding) were formally assessed for bias. Candidate biomarkers were identified by an expert multistakeholder group using a majority voting consensus exercise and mapped to relevant cellular and molecular pathways. RESULTS: Of 71 included studies (67 studying effectiveness outcomes and eight safety outcomes; four studied both), most reported genomic or proteomic biomarkers associated with response to biologics (48 studies). Methodological or reporting limitations frequently compromised the interpretation of findings, including inadequate control for key covariates, lack of adjustment for multiple testing, and selective outcome reporting. We identified candidate biomarkers of efficacy to tumour necrosis factor inhibitors [variation in CARD14, CDKAL1, IL1B, IL12B and IL17RA loci, and lipopolysaccharide-induced phosphorylation of nuclear factor (NF)-κB in type 2 dendritic cells] and ustekinumab (HLA-C*06:02 and variation in an IL1B locus). None were supported by sufficient evidence for clinical use without further validation studies. Candidate biomarkers were found to be involved in the immune cellular crosstalk implicated in psoriasis pathogenesis, most notably antigen presentation, T helper (Th)17 cell differentiation, positive regulation of NF-κB, and Th17 cell activation. CONCLUSIONS: This comprehensive catalogue provides a key resource for researchers and reveals a diverse range of biomarker types and outcomes in the included studies. The candidate biomarkers identified require further evaluation in methodologically robust studies to establish potential clinical utility. Future studies should aim to address the common methodological limitations highlighted in this review to expedite discovery and validation of biomarkers for clinical use. What is already known about this topic? Responses to the systemic treatments commonly used to treat psoriasis vary. Biomarkers that accurately predict effectiveness and safety would enable targeted treatment selection, improved patient outcomes and more cost-effective healthcare. What does this study add? This review provides a comprehensive catalogue of investigated biomarkers of systemic treatment response in psoriasis. A diverse range of biomarker types and outcomes was found in the included studies, serving as a key resource for the translational research community.

Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder.

ATP synthase, H+ transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F1FO ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.

The neurotrophic factor receptor RET drives haematopoietic stem cell survival and function.

Haematopoiesis is a developmental cascade that generates all blood cell lineages in health and disease. This process relies on quiescent haematopoietic stem cells capable of differentiating, self renewing and expanding upon physiological demand. However, the mechanisms that regulate haematopoietic stem cell homeostasis and function remain largely unknown. Here we show that the neurotrophic factor receptor RET (rearranged during transfection) drives haematopoietic stem cell survival, expansion and function. We find that haematopoietic stem cells express RET and that its neurotrophic factor partners are produced in the haematopoietic stem cell environment. Ablation of Ret leads to impaired survival and reduced numbers of haematopoietic stem cells with normal differentiation potential, but loss of cell-autonomous stress response and reconstitution potential. Strikingly, RET signals provide haematopoietic stem cells with critical Bcl2 and Bcl2l1 surviving cues, downstream of p38 mitogen-activated protein (MAP) kinase and cyclic-AMP-response element binding protein (CREB) activation. Accordingly, enforced expression of RET downstream targets, Bcl2 or Bcl2l1, is sufficient to restore the activity of Ret null progenitors in vivo. Activation of RET results in improved haematopoietic stem cell survival, expansion and in vivo transplantation efficiency. Remarkably, human cord-blood progenitor expansion and transplantation is also improved by neurotrophic factors, opening the way for exploration of RET agonists in human haematopoietic stem cell transplantation. Our work shows that neurotrophic factors are novel components of the haematopoietic stem cell microenvironment, revealing that haematopoietic stem cells and neurons are regulated by similar signals.

Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity.

The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Dynamic PIN-FORMED auxin efflux carrier phosphorylation at the plasma membrane controls auxin efflux-dependent growth.

The directional distribution of the phytohormone auxin is essential for plant development. Directional auxin transport is mediated by the polarly distributed PIN-FORMED (PIN) auxin efflux carriers. We have previously shown that efficient PIN1-mediated auxin efflux requires activation through phosphorylation at the four serines S1-S4 in Arabidopsis thaliana The Brefeldin A (BFA)-sensitive D6 PROTEIN KINASE (D6PK) and the BFA-insensitive PINOID (PID) phosphorylate and activate PIN1 through phosphorylation at all four phosphosites. PID, but not D6PK, can also induce PIN1 polarity shifts, seemingly through phosphorylation at S1-S3. The differential effects of D6PK and PID on PIN1 polarity had so far been attributed to their differential phosphosite preference for the four PIN1 phosphosites. We have mapped PIN1 phosphorylation at S1-S4 in situ using phosphosite-specific antibodies. We detected phosphorylation at PIN1 phosphosites at the basal (rootward) as well as the apical (shootward) plasma membrane in different root cell types, in embryos, and shoot apical meristems. Thereby, PIN1 phosphorylation at all phosphosites generally followed the predominant PIN1 distribution but was not restricted to specific polar sides of the cells. PIN1 phosphorylation at the basal and apical plasma membrane was differentially sensitive to BFA treatments, suggesting the involvement of different protein kinases or trafficking mechanisms in PIN1 phosphorylation control. We conclude that phosphosite preferences are not sufficient to explain the differential effects of D6PK and PID on PIN1 polarity, and suggest that a more complex model is needed to explain the effects of PID.

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number.

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.

Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases.

The Arabidopsis SR45 Splicing Factor, a Negative Regulator of Sugar Signaling, Modulates SNF1-Related Protein Kinase 1 Stability.

The ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We previously reported that the SR45 splicing factor negatively regulates glucose signaling during early seedling development in Arabidopsis thaliana Here, we show that under glucose-fed conditions, the Arabidopsis sr45-1 loss-of-function mutant contains higher amounts of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) despite unaffected SnRK1 transcript levels. In agreement, marker genes for SnRK1 activity are upregulated in sr45-1 plants, and the glucose hypersensitivity of sr45-1 is attenuated by disruption of the SnRK1 gene. Using a high-resolution RT-PCR panel, we found that the sr45-1 mutation broadly targets alternative splicing in vivo, including that of the SR45 pre-mRNA itself. Importantly, the enhanced SnRK1 levels in sr45-1 are suppressed by a proteasome inhibitor, indicating that SR45 promotes targeting of the SnRK1 protein for proteasomal destruction. Finally, we demonstrate that SR45 regulates alternative splicing of the Arabidopsis 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of degradation of the SnRK1 energy sensor in response to sugars.

TRAP1 regulates autophagy in lung cancer cells.

BACKGROUND: Expression of TRAP1, a member of the HSP90 chaperone family, has been implicated in tumour protective effects, based on its differential mitochondrial localization and function. DESIGN: This work was designed to provide new insights into the pathways involved in TRAP1-provided cytoprotection on NSCLC. For this, TRAP1-depleted A549 human NSCLC cells and MRC-5 normal lung fibroblasts were produced using a siRNA approach and main cellular quality control mechanisms were investigated. RESULTS: TRAP1-depleted A549 cells displayed decreased cell viability likely due to impaired mitochondrial function including decreased ATP/AMP ratio, oxygen consumption and membrane potential, as well as increased apoptotic indicators. Furthermore, the negative impact of TRAP1 depletion on mitochondrial function was not observed in normal MRC-5 lung cells, which might be due to the differential intracellular localization of the chaperone in tumour versus normal cells. Additionally, A549 TRAP1-depleted cells showed increased autophagic flux. Functionally, autophagy inhibition resulted in decreased cell viability in both TRAP1-expressing and TRAP1-depleted tumour cells with minor effects on MRC-5 cells. Conversely, autophagy stimulation decreased cell viability of both A549 and MRC-5 TRAP1-expressing cells while in A549 TRAP1-depleted cells, increased autophagy augmented viability. CONCLUSIONS: Our results show that even though TRAP1 depletion affects both normal MRC-5 and tumour A549 cell proliferation, inhibition of autophagy per se led to a decrease in tumour cell mass, while having a reduced effect on the normal cell line. The strategy of targeting TRAP1 in NSCLC shows future potential therapeutic applications.

Novel GFM2 variants associated with early-onset neurological presentations of mitochondrial disease and impaired expression of OXPHOS subunits.

Mitochondrial diseases are characterised by clinical, molecular and functional heterogeneity, reflecting their bi-genomic control. The nuclear gene GFM2 encodes mtEFG2, a protein with an essential role during the termination stage of mitochondrial translation. We present here two unrelated patients harbouring different and previously unreported compound heterozygous (c.569G>A, p.(Arg190Gln); c.636delA, p.(Glu213Argfs*3)) and homozygous (c.275A>C, p.(Tyr92Ser)) recessive variants in GFM2 identified by whole exome sequencing (WES) together with histochemical and biochemical findings to support the diagnoses of pathological GFM2 variants in each case. Both patients presented similarly in early childhood with global developmental delay, raised CSF lactate and abnormalities on cranial MRI. Sanger sequencing of familial samples confirmed the segregation of bi-allelic GFM2 variants with disease, while investigations into steady-state mitochondrial protein levels revealed respiratory chain subunit defects and loss of mtEFG2 protein in muscle. These data demonstrate the effects of defective mtEFG2 function, caused by previously unreported variants, confirming pathogenicity and expanding the clinical phenotypes associated with GFM2 variants.