ABSTRACT
Sanfilippo syndrome is a fatal childhood neurodegenerative disorder involving neuroinflammation among multiple pathologies. We hypothesized that anakinra, a recombinant interleukin-1 receptor antagonist, could improve neurobehavioral and functional symptoms owing to its capacity to treat neuroinflammation. This phase 1/2 trial aimed to test the safety, tolerability and effects of anakinra on neurobehavioral, functional and quality-of-life outcomes in patients and their caregivers. The primary outcome was the percent of participants requiring a dose increase at week 8 or week 16. Secondary efficacy outcomes included a multi-domain responder index (MDRI). Twenty-three participants (6-26 years of age) were enrolled. Twenty continued treatment to week 8, and 15 (75%) required an increased dose at week 8 or week 16. There was an improvement in at least one domain in the MDRI in 18 of 21 (86%) at week 8 and in 15 of 16 (94%) at week 36. Seven participants withdrew (intolerability of daily injections and lost to follow-up) before week 36. Adverse events occurred in 22 of 23 (96%) participants, most commonly mild injection site reactions. No serious adverse events were related to anakinra. In conclusion, anakinra was safe and associated with improved neurobehavioral and functional outcomes, supporting continued investigation of anakinra in Sanfilippo syndrome and other mucopolysaccharidoses. ClinicalTrials.gov identifier: NCT04018755 .
ABSTRACT
p97/VCP, a hexametric member of the AAA-ATPase superfamily, has been associated with a wide range of cellular protein pathways, such as proteasomal degradation, the unfolding of polyubiquitinated proteins, and autophagosome maturation. Autosomal dominant p97/VCP mutations cause a rare hereditary multisystem disorder called IBMPFD/ALS (Inclusion Body Myopathy with Paget's Disease and Frontotemporal Dementia/Amyotrophic Lateral Sclerosis), characterized by progressive weakness and subsequent atrophy of skeletal muscles, and impacting bones and brains, such as Parkinson's disease, Lewy body disease, Huntington's disease, and amyotrophic lateral ALS. Among all disease-causing mutations, Arginine 155 to Histidine (R155H/+) was reported to be the most common one, affecting over 50% of IBMPFD patients, resulting in disabling muscle weakness, which might eventually be life-threatening due to cardiac and respiratory muscle involvement. Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to study pathology's underlying molecular mechanism, perform drug screening, and investigate regeneration. Using R155H/+ patients' fibroblasts, we generated IPS cells and corrected the mutation (Histidine to Arginine, H155R) to generate isogenic control cells before differentiating them into myotubes. The further proteomic analysis allowed us to identify differentially expressed proteins associated with the R155H mutation. Our results showed that R155H/+ cells were associated with dysregulated expression of several proteins involved in skeletal muscle function, cytoskeleton organization, cell signaling, intracellular organelles organization and function, cell junction, and cell adhesion. Our findings provide molecular evidence of dysfunctional protein expression in R155H/+ myotubes and offer new therapeutic targets for treating IBMPFD/ALS.
ABSTRACT
Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB) is a recessive genetic disorder that severely affects the brain due to a deficiency in the enzyme α-N-acetylglucosaminidase (NAGLU), leading to intra-lysosomal accumulation of partially degraded heparan sulfate. There are no effective treatments for this disorder. In this project, we carried out an ex vivo correction of neural stem cells derived from Naglu -/- mice (iNSCs) induced pluripotent stem cells (iPSC) using a modified enzyme in which human NAGLU is fused to an insulin-like growth factor II receptor binding peptide in order to improve enzyme uptake. After brain transplantation of corrected iNSCs into Naglu -/- mice and long-term evaluation of their impact, we successfully detected NAGLU-IGFII activity in all transplanted animals. We found decreased lysosomal accumulation and reduced astrocytosis and microglial activation throughout transplanted brains. We also identified a novel neuropathological phenotype in untreated Naglu -/- brains with decreased levels of the neuronal marker Map2 and accumulation of synaptophysin-positive aggregates. Upon transplantation, we restored levels of Map2 expression and significantly reduced formation of synaptophysin-positive aggregates. Our findings suggest that genetically engineered iNSCs can be used to effectively deliver the missing enzyme to the brain and treat Sanfilippo type B-associated neuropathology.
ABSTRACT
The extracellular matrix (ECM) provides essential cues to promote endothelial specification during tissue development in vivo; correspondingly, ECM is considered essential for endothelial differentiation outside of the body. However, systematic studies to assess the precise contribution of individual ECM proteins to endothelial differentiation have not been conducted. Further, the multi-component nature of differentiation protocols makes it challenging to study the underlying mechanisms by which the ECM contributes to cell fate. In this study, we determined that Laminin 411 alone increases endothelial differentiation of induced pluripotent stem cells over collagen I or Matrigel. The effect of ECM was shown to be independent of vascular endothelial growth factor (VEGF) binding capacity. We also show that ECM-guided endothelial differentiation is dependent on activation of focal adhesion kinase (FAK), integrin-linked kinase (ILK), Notch, and ß-catenin pathways. Our results indicate that ECM contributes to endothelial differentiation through multiple avenues, which converge at the expression of active ß-catenin.
Subject(s)
Laminin , beta Catenin , Extracellular Matrix/metabolism , Laminin/metabolism , Signal Transduction/physiology , Vascular Endothelial Growth Factor A/metabolism , beta Catenin/metabolismABSTRACT
Ataxia Telangiectasia (A-T) and Ataxia with Ocular Apraxia Type 1 (AOA1) are devastating neurological disorders caused by null mutations in the genome stability genes, A-T mutated (ATM) and Aprataxin (APTX), respectively. Our mechanistic understanding and therapeutic repertoire for treating these disorders are severely lacking, in large part due to the failure of prior animal models with similar null mutations to recapitulate the characteristic loss of motor coordination (i.e., ataxia) and associated cerebellar defects. By increasing genotoxic stress through the insertion of null mutations in both the Atm (nonsense) and Aptx (knockout) genes in the same animal, we have generated a novel mouse model that for the first time develops a progressively severe ataxic phenotype associated with atrophy of the cerebellar molecular layer. We find biophysical properties of cerebellar Purkinje neurons (PNs) are significantly perturbed (e.g., reduced membrane capacitance, lower action potential [AP] thresholds, etc.), while properties of synaptic inputs remain largely unchanged. These perturbations significantly alter PN neural activity, including a progressive reduction in spontaneous AP firing frequency that correlates with both cerebellar atrophy and ataxia over the animal's first year of life. Double mutant mice also exhibit a high predisposition to developing cancer (thymomas) and immune abnormalities (impaired early thymocyte development and T-cell maturation), symptoms characteristic of A-T. Finally, by inserting a clinically relevant nonsense-type null mutation in Atm, we demonstrate that Small Molecule Read-Through (SMRT) compounds can restore ATM production, indicating their potential as a future A-T therapeutic.
Subject(s)
Ataxia Telangiectasia/genetics , Atrophy/physiopathology , Cerebellum/pathology , Codon, Nonsense/genetics , Purkinje Cells/metabolism , Animals , Ataxia Telangiectasia/physiopathology , Atrophy/genetics , Disease Models, Animal , Female , Male , MiceABSTRACT
Mucopolysaccharidoses III (MPS III, Sanfilippo syndrome) is a subtype of the Mucopolysaccharidoses (MPS), a group of inherited lysosomal disorders caused by a deficiency of lysosomal enzymes responsible for catabolizing glycosaminoglycans (GAGs). Although MPS III is rare, MPS diseases as a group are relatively frequent with an overall incidence of approximately 1 in 20,000 - 25,000 births. MPS III are paediatric diseases, which cause learning difficulties, behavioural disorders and dementia, as well as skeletal deformities and ultimately result in premature death. There are currently no approved treatments for MPS III, but a number of therapeutic approaches are under development. In the past 30 years, research using cellular and animal models have led to clinical trials involving enzyme replacement therapy (ERT), substrate reduction therapy (SRT) and gene therapy, while stem cells approaches remain at the pre-clinical stage. Although safety and clinical efficacy in animal models have shown promise, the results of clinical trials have proved costly and shown limited therapeutic effects. In this review, we describe the most recent results from clinical trials. While ERT and gene therapy are the most developed therapies for MPS III, we highlight the work that needs to be done to bring us closer to a real treatment for these devastating diseases.
ABSTRACT
The article Tet3 regulates cellular identity and DNA methylation in neural progenitor cells, written by Miguel R. Branco and C. Joana Marques, was originally published electronically on the publisher's internet portal.
ABSTRACT
TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.
Subject(s)
Cell Differentiation/genetics , DNA Methylation/genetics , Dioxygenases/genetics , Neural Stem Cells/metabolism , 5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Brain/growth & development , Brain/metabolism , DNA-Binding Proteins/genetics , Gene Knockdown Techniques , Genomic Imprinting/genetics , Humans , Mice , Mouse Embryonic Stem Cells/metabolism , Neurogenesis/genetics , Neurons/metabolism , Promoter Regions, Genetic/genetics , RNA-Binding Proteins/geneticsABSTRACT
Patient-derived induced pluripotent stem cells (iPSCs) have become a promising resource for exploring genetics of complex diseases, discovering new drugs, and advancing regenerative medicine. Increasingly, laboratories are creating their own banks of iPSCs derived from diverse donors. However, there are not yet standardized guidelines for qualifying these cell lines, i.e., distinguishing between bona fide human iPSCs, somatic cells, and imperfectly reprogrammed cells. Here, we report the establishment of a panel of 30 iPSCs from CD34+ peripheral blood mononuclear cells, of which 10 were further differentiated in vitro into all three germ layers. We characterized these different cell types with commonly used pluripotent and lineage specific markers, and showed that NES, TUBB3, and OTX2 cannot be reliably used as ectoderm differentiation markers. Our work highlights the importance of marker selection in iPSC authentication, and the need for the field to establish definitive standard assays.
Subject(s)
Antigens, Differentiation/metabolism , Biomarkers/metabolism , Cell Differentiation , Ectoderm/metabolism , Induced Pluripotent Stem Cells/cytology , Leukocytes, Mononuclear/metabolism , Cells, Cultured , Ectoderm/cytology , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/cytologyABSTRACT
Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB [MPS IIIB]) is a lysosomal storage disorder primarily affecting the brain that is caused by a deficiency in the enzyme α-N-acetylglucosaminidase (NAGLU), leading to intralysosomal accumulation of heparan sulfate. There are currently no treatments for this disorder. Here we report that, ex vivo, lentiviral correction of Naglu-/- neural stem cells derived from Naglu-/- mice (iNSCs) corrected their lysosomal pathology and allowed them to secrete a functional NAGLU enzyme that could be taken up by deficient cells. Following long-term transplantation of these corrected iNSCs into Naglu-/- mice, we detected NAGLU activity in the majority of engrafted animals. Successfully transplanted Naglu-/- mice showed a significant decrease in storage material, a reduction in astrocyte activation, and complete prevention of microglial activation within the area of engrafted cells and neighboring regions, with beneficial effects extending partway along the rostrocaudal axis of the brain. Our results demonstrate long-term engraftment of iNSCs in the brain that are capable of cross-correcting pathology in Naglu-/- mice. Our findings suggest that genetically engineered iNSCs could potentially be used to deliver enzymes and treat MPS IIIB.
ABSTRACT
BACKGROUND: Anti-metabolites are less-myelosuppressive than DNA-damaging anticancer drugs and may be useful against brain tumors. MATERIALS AND METHODS: We evaluated the asparagine/glutamine-deaminating agent Erwinaze with/without temozolomide against brain tumor cells and mouse medulloblastomas. RESULTS: Erwinaze treatment of cell lines and neurospheres led to dose-dependent reductions of cells (reversible by L-glutamine), with half maximal inhibitory concentrations (IC50s) of 0.12->10 IU/ml. Erwinaze at <1 IU/ml reduced temozolomide IC50s by 3.6- to 13-fold (300-1,200 µM to 40-330 µM). Seven-week-old SMO/SMO mice treated with Erwinaze (regardless of temozolomide treatment) had better survival 11 weeks post-therapy, compared to those not treated with Erwinaze (81.25% vs. 46.15, p=0.08). Temozolomide-treated mice developed 10% weight loss, impairing survival. All 16 mice treated with temozolomide (regardless of Erwinaze treatment) succumbed by 40-weeks of age, whereas 5/8 animals treated with Erwinaze alone and 2/6 controls survived (p=0.035). CONCLUSION: Erwinaze enhances cytotoxicity of temozolomide in vitro, and improves survival in SMO/SMO mice, likely by reducing cerebrospinal fluid glutamine. Temozolomide-associated toxicity prevented demonstration of any potential combinatorial advantage with Erwinaze in vivo.
Subject(s)
Antineoplastic Agents/therapeutic use , Asparaginase/therapeutic use , Brain Neoplasms/drug therapy , Cerebellar Neoplasms/drug therapy , Glioblastoma/drug therapy , Medulloblastoma/drug therapy , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Asparaginase/administration & dosage , Brain Neoplasms/pathology , Cell Line, Tumor , Culture Media, Serum-Free , Dacarbazine/administration & dosage , Dacarbazine/analogs & derivatives , Dickeya chrysanthemi/enzymology , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Drug Synergism , Glioblastoma/pathology , Glioma/drug therapy , Glioma/pathology , Glutamine/pharmacology , Humans , Mice , Mice, Inbred C57BL , Radiation Tolerance , Spheroids, Cellular/drug effects , Temozolomide , Xenograft Model Antitumor AssaysABSTRACT
Antibodies against recombinant proteins can significantly reduce their effectiveness in unanticipated ways. We evaluated the humoral response of mice with the lysosomal storage disease mucopolysaccharidosis type I treated with weekly intravenous recombinant human alpha-l-iduronidase (rhIDU). Unlike patients, the majority of whom develop antibodies to recombinant human alpha-l-iduronidase, only approximately half of the treated mice developed antibodies against recombinant human alpha-l-iduronidase and levels were low. Serum from antibody-positive mice inhibited uptake of recombinant human alpha-l-iduronidase into human fibroblasts by partial inhibition compared to control serum. Tissue and cellular distributions of rhIDU were altered in antibody-positive mice compared to either antibody-negative or naive mice, with significantly less recombinant human alpha-l-iduronidase activity in the heart and kidney in antibody-positive mice. In the liver, recombinant human alpha-l-iduronidase was preferentially found in sinusoidal cells rather than in hepatocytes in antibody-positive mice. Antibodies against recombinant human alpha-l-iduronidase enhanced uptake of recombinant human alpha-l-iduronidase into macrophages obtained from MPS I mice. Collectively, these results imply that a humoral immune response against a therapeutic protein can shift its distribution preferentially into macrophage-lineage cells, causing decreased availability of the protein to the cells that are its therapeutic targets.
ABSTRACT
Previous retroviral and knock-in approaches to model human t(11;19)+ acute mixed-lineage leukemia in mice resulted in myeloproliferation and acute myeloid leukemia not fully recapitulating the human disease. The authors established a doxycycline (DOX)-inducible transgenic mouse model "iMLL-ENL" in which induction in long-term hematopoietic stem cells, lymphoid primed multipotent progenitor cells, multipotent progenitors (MPP4) but not in more committed myeloid granulocyte-macrophage progenitors led to a fully reversible acute leukemia expressing myeloid and B-cell markers. iMLL-ENL leukemic cells generally expressed lower MLL-ENL mRNA than those obtained after retroviral transduction. Disease induction was associated with iMLL-ENL levels exceeding the endogenous Mll1 at mRNA and protein levels. In leukemic cells from t(11;19)+ leukemia patients, MLL-ENL mRNA also exceeded the endogenous MLL1 levels suggesting a critical threshold for transformation. Expression profiling of iMLL-ENL acute leukemia revealed gene signatures that segregated t(11;19)+ leukemia patients from those without an MLL translocation. Importantly, B220+ iMLL-ENL leukemic cells showed a higher in vivo leukemia initiation potential than coexisting B220- cells. Collectively, characterization of a novel transgenic mouse model indicates that the cell-of-origin and the fusion gene expression levels are both critical determinants for MLL-ENL-driven acute leukemia.
ABSTRACT
Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT.
Subject(s)
Endothelial Cells/metabolism , Hematopoiesis/physiology , Homeodomain Proteins/metabolism , Receptors, Notch/metabolism , Signal Transduction/physiology , Animals , Down-Regulation/physiology , Endothelial Cells/cytology , Homeodomain Proteins/genetics , Jagged-1 Protein/biosynthesis , Jagged-1 Protein/genetics , Mice , Receptors, Notch/geneticsABSTRACT
The hematopoietic stem cell-enriched miR-125 family microRNAs (miRNAs) are critical regulators of hematopoiesis. Overexpression of miR-125a or miR-125b is frequent in human acute myeloid leukemia (AML), and the overexpression of these miRNAs in mice leads to expansion of hematopoietic stem cells accompanied by perturbed hematopoiesis with mostly myeloproliferative phenotypes. However, whether and how miR-125 family miRNAs cooperate with known AML oncogenes in vivo, and how the resultant leukemia is dependent on miR-125 overexpression, are not well understood. We modeled the frequent co-occurrence of miR-125b overexpression and MLL translocations by examining functional cooperation between miR-125b and MLL-AF9 By generating a knock-in mouse model in which miR-125b overexpression is controlled by doxycycline induction, we demonstrated that miR-125b significantly enhances MLL-AF9-driven AML in vivo, and the resultant leukemia is partially dependent on continued overexpression of miR-125b Surprisingly, miR-125b promotes AML cell expansion and suppresses apoptosis involving a non-cell-intrinsic mechanism. MiR-125b expression enhances VEGFA expression and production from leukemia cells, in part by suppressing TET2 Recombinant VEGFA recapitulates the leukemia-promoting effects of miR-125b, whereas knockdown of VEGFA or inhibition of VEGF receptor 2 abolishes the effects of miR-125b In addition, significant correlation between miR-125b and VEGFA expression is observed in human AMLs. Our data reveal cooperative and dependent relationships between miR-125b and the MLL oncogene in AML leukemogenesis, and demonstrate a miR-125b-TET2-VEGFA pathway in mediating non-cell-intrinsic leukemia-promoting effects by an oncogenic miRNA.
Subject(s)
Leukemia, Myeloid, Acute/etiology , MicroRNAs/physiology , Oncogene Proteins, Fusion/physiology , Vascular Endothelial Growth Factor A/physiology , Animals , Apoptosis , Cell Proliferation , Gene Expression Regulation, Leukemic , Gene Knock-In Techniques , Hematopoiesis , Hematopoietic Stem Cells , Leukemia, Myeloid, Acute/metabolism , Mice , Myeloid-Lymphoid Leukemia Protein/geneticsABSTRACT
OBJECTIVE: Although intrauterine nutritional stress is known to result in offspring obesity and the metabolic phenotype, the underlying cellular/molecular mechanisms remain incompletely understood. We tested the hypothesis that compared with the controls, the bone marrow-derived mesenchymal stem cells (BMSCs) of the intrauterine growth-restricted (IUGR) offspring exhibit a more adipogenic phenotype. METHODS: A well-established rat model of maternal food restriction (MFR), that is, 50% global caloric restriction during the later-half of pregnancy and ad libitum diet following birth that is known to result in an obese offspring with a metabolic phenotype was used. BMSCs at 3 weeks of age were isolated, and then molecularly and functionally profiled. RESULTS: BMSCs of the intrauterine nutritionally-restricted offspring demonstrated an increased proliferation and an enhanced adipogenic molecular profile at miRNA, mRNA and protein levels, with an overall up-regulated PPARγ (miR-30d, miR-103, PPARγ, C/EPBα, ADRP, LPL, SREBP1), but down-regulated Wnt (LRP5, LEF-1, ß-catenin, ZNF521 and RUNX2) signaling profile. Following adipogenic induction, compared with the control BMSCs, the already up-regulated adipogenic profile of the MFR BMSCs, showed a further increased adipogenic response. CONCLUSIONS: Markedly enhanced adipogenic molecular profile and increased cell proliferation of MFR BMSCs suggest a possible novel cellular/mechanistic link between the intrauterine nutritional stress and offspring metabolic phenotype. This provides new potential predictive and therapeutic targets against these conditions in the IUGR offspring.
Subject(s)
Adipogenesis/physiology , Fetal Growth Retardation/pathology , Maternal Nutritional Physiological Phenomena/physiology , Mesenchymal Stem Cells/metabolism , Peroxisome Proliferator-Activated Receptors/physiology , Wnt Signaling Pathway/physiology , Animals , Animals, Newborn , Caloric Restriction , Cell Differentiation , Disease Models, Animal , Down-Regulation , Female , Fetal Growth Retardation/genetics , Maternal Nutritional Physiological Phenomena/genetics , MicroRNAs , Phenotype , Pregnancy , RNA, Messenger , Rats , Rats, Sprague-Dawley , Up-RegulationABSTRACT
To address the impact of cellular origin on acute myeloid leukemia (AML), we generated an inducible transgenic mouse model for MLL-AF9-driven leukemia. MLL-AF9 expression in long-term hematopoietic stem cells (LT-HSC) in vitro resulted in dispersed clonogenic growth and expression of genes involved in migration and invasion. In vivo, 20% LT-HSC-derived AML were particularly aggressive with extensive tissue infiltration, chemoresistance, and expressed genes related to epithelial-mesenchymal transition (EMT) in solid cancers. Knockdown of the EMT regulator ZEB1 significantly reduced leukemic blast invasion. By classifying mouse and human leukemias according to Evi1/EVI1 and Erg/ERG expression, reflecting aggressiveness and cell of origin, and performing comparative transcriptomics, we identified several EMT-related genes that were significantly associated with poor overall survival of AML patients.
Subject(s)
Epithelial-Mesenchymal Transition , Hematopoietic Stem Cells/cytology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Animals , Drug Resistance, Neoplasm , Gene Expression Profiling/methods , Gene Expression Regulation, Leukemic , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism , Mice , Mice, Transgenic , Neoplasm Invasiveness , Neoplasms, Experimental , Prognosis , Tumor Cells, Cultured , Zinc Finger E-box-Binding Homeobox 1/geneticsABSTRACT
Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget's disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (kcat/Km) of p97 by 11-fold. Whereas both p37 and p47 decrease the Km of ATP in p97, p37 increases the kcat of p97. In contrast, regulation by p47 is biphasic, with decreased kcat at low levels but increased kcat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69-92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactor-regulated communication may contribute to p97-associated disease pathogenesis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Mutation , Adenosine Triphosphate/metabolism , Autophagy , Bone Diseases/metabolism , Cell Line, Tumor , Chromatography, Gel , Golgi Apparatus , Homeostasis , Humans , Muscular Diseases/metabolism , Neurodegenerative Diseases/metabolism , Phenotype , Protein Structure, Tertiary , Surface Plasmon Resonance , Valosin Containing ProteinABSTRACT
Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease.