ABSTRACT
Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)-engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.
Subject(s)
Proliferating Cell Nuclear Antigen/metabolism , Protein Engineering/methods , Proteolysis , Ubiquitin-Protein Ligases/genetics , Binding Sites , HEK293 Cells , Humans , Molecular Targeted Therapy/methods , Proliferating Cell Nuclear Antigen/chemistry , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.
Subject(s)
Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/physiology , Developmental Disabilities/genetics , Growth Disorders/genetics , Mutation , Spine/abnormalities , Spine/pathology , Animals , Cell Cycle , Cell Proliferation , Cells, Cultured , Child , Child, Preschool , Cilia/metabolism , Cilia/pathology , Developmental Disabilities/pathology , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Growth Disorders/pathology , Humans , Infant , Male , Mice , Mice, Knockout , Pedigree , Phosphorylation , Signal Transduction , Spine/metabolismABSTRACT
Stapled α-helical peptides represent an emerging superclass of macrocyclic molecules with drug-like properties, including high-affinity target binding, protease resistance, and membrane permeability. As a model system for probing the chemical space available for optimizing these properties, we focused on dual Mdm2/MdmX antagonist stapled peptides related to the p53 N-terminus. Specifically, we first generated a library of ATSP-7041 (Chang et al., 2013) analogs iteratively modified by L-Ala and D-amino acids. Single L-Ala substitutions beyond the Mdm2/(X) binding interfacial residues (i.e., Phe3, Trp7, and Cba10) had minimal effects on target binding, α-helical content, and cellular activity. Similar binding affinities and cellular activities were noted at non-interfacial positions when the template residues were substituted with their d-amino acid counterparts, despite the fact that d-amino acid residues typically 'break' right-handed α-helices. d-amino acid substitutions at the interfacial residues Phe3 and Cba10 resulted in the expected decreases in binding affinity and cellular activity. Surprisingly, substitution at the remaining interfacial position with its d-amino acid equivalent (i.e., Trp7 to d-Trp7) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp7-modified peptide was determined and revealed that the indole side chain was able to interact optimally with its Mdm2 binding site by a slight global re-orientation of the stapled peptide. To further investigate the comparative effects of d-amino acid substitutions we used linear analogs of ATSP-7041, where we replaced the stapling amino acids by Aib (i.e., R84 to Aib4 and S511 to Aib11) to retain the helix-inducing properties of α-methylation. The resultant analog sequence Ac-Leu-Thr-Phe-Aib-Glu-Tyr-Trp-Gln-Leu-Cba-Aib-Ser-Ala-Ala-NH2 exhibited high-affinity target binding (Mdm2 Kd = 43 nM) and significant α-helicity in circular dichroism studies. Relative to this linear ATSP-7041 analog, several d-amino acid substitutions at Mdm2(X) non-binding residues (e.g., d-Glu5, d-Gln8, and d-Leu9) demonstrated decreased binding and α-helicity. Importantly, circular dichroism (CD) spectroscopy showed that although helicity was indeed disrupted by d-amino acids in linear versions of our template sequence, stapled molecules tolerated these residues well. Further studies on stapled peptides incorporating N-methylated amino acids, l-Pro, or Gly substitutions showed that despite some positional dependence, these helix-breaking residues were also generally tolerated in terms of secondary structure, binding affinity, and cellular activity. Overall, macrocyclization by hydrocarbon stapling appears to overcome the destabilization of α-helicity by helix breaking residues and, in the specific case of d-Trp7-modification, a highly potent ATSP-7041 analog (Mdm2 Kd = 30 nM; cellular EC50 = 600 nM) was identified. Our findings provide incentive for future studies to expand the chemical diversity of macrocyclic α-helical peptides (e.g., d-amino acid modifications) to explore their biophysical properties and cellular permeability. Indeed, using the library of 50 peptides generated in this study, a good correlation between cellular permeability and lipophilicity was observed.
Subject(s)
Amino Acids/chemistry , Cell-Penetrating Peptides/chemistry , Peptide Fragments/chemistry , Protein Conformation , Amino Acid Sequence/genetics , Amino Acid Substitution/genetics , Amino Acids/chemical synthesis , Cell-Penetrating Peptides/chemical synthesis , Cell-Penetrating Peptides/genetics , Cell-Penetrating Peptides/pharmacology , Circular Dichroism , Dipeptides/chemistry , Humans , Oligopeptides/chemistry , Peptides, Cyclic/pharmacology , Permeability/drug effects , Protein Structure, Secondary , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/geneticsABSTRACT
Macrocyclic α-helical peptides have emerged as a compelling new therapeutic modality to tackle targets confined to the intracellular compartment. Within the scope of hydrocarbon-stapling there has been significant progress to date, including the first stapled α-helical peptide to enter into clinical trials. The principal design concept of stapled α-helical peptides is to mimic a cognate (protein) ligand relative to binding its target via an α-helical interface. However, it was the proclivity of such stapled α-helical peptides to exhibit cell permeability and proteolytic stability that underscored their promise as unique macrocyclic peptide drugs for intracellular targets. This perspective highlights key learnings as well as challenges in basic research with respect to structure-based design, innovative chemistry, cell permeability and proteolytic stability that are essential to fulfill the promise of stapled α-helical peptide drug development.
Subject(s)
Drug Discovery/methods , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Peptides/chemistry , Peptides/pharmacology , Animals , Humans , Macrocyclic Compounds/pharmacokinetics , Models, Molecular , Peptides/pharmacokinetics , Protein Conformation, alpha-HelicalABSTRACT
Cyclin-dependent kinases (Cdks) are serine/threonine kinases and their catalytic activities are modulated by interactions with cyclins and Cdk inhibitors (CKIs). Close cooperation between this trio is necessary for ensuring orderly progression through the cell cycle. In addition to their well-established function in cell cycle control, it is becoming increasingly apparent that mammalian Cdks, cyclins and CKIs play indispensable roles in processes such as transcription, epigenetic regulation, metabolism, stem cell self-renewal, neuronal functions and spermatogenesis. Even more remarkably, they can accomplish some of these tasks individually, without the need for Cdk/cyclin complex formation or kinase activity. In this Review, we discuss the latest revelations about Cdks, cyclins and CKIs with the goal of showcasing their functional diversity beyond cell cycle regulation and their impact on development and disease in mammals.
Subject(s)
Cell Cycle Checkpoints/physiology , Cyclin-Dependent Kinase Inhibitor Proteins/physiology , Cyclin-Dependent Kinases/physiology , Cyclins/physiology , Amino Acid Sequence , Animals , Cell Cycle Checkpoints/genetics , Cyclin-Dependent Kinase Inhibitor Proteins/genetics , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/genetics , Cyclins/genetics , DNA Repair , Epigenesis, Genetic , Humans , Male , Molecular Sequence Data , Neurons/physiology , Proteolysis , Spermatogenesis , Stem Cells/cytology , Stem Cells/metabolism , Transcription, GeneticABSTRACT
Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well-controlled cell model systems. We have developed a first nonintegration-reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high-resolution whole genome CGH-array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high-content microscopic analysis. Early differentiation shows an imbalance of the lineage-specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC-derived neurons show increased production of amyloid peptide-containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21-derived neurons show significantly higher number of DNA double-strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21.
Subject(s)
Aging/physiology , Cell Differentiation/physiology , Down Syndrome/genetics , Induced Pluripotent Stem Cells/cytology , Neurons/cytology , Animals , Cells, Cultured , Fibroblasts/cytology , Humans , Mitochondria/geneticsABSTRACT
Mouse knockouts of Cdk2 and Cdk4 are individually viable whereas the double knockouts are embryonic lethal due to heart defects, and this precludes the investigation of their overlapping roles in definitive hematopoiesis. Here we use a conditional knockout mouse model to investigate the effect of combined loss of Cdk2 and Cdk4 in hematopoietic cells. Cdk2(fl/fl)Cdk4(-/-)vavCre mice are viable but displayed a significant increase in erythrocyte size. Cdk2(fl/fl)Cdk4(-/-)vavCre mouse bone marrow exhibited reduced phosphorylation of the retinoblastoma protein and reduced expression of E2F target genes such as cyclin A2 and Cdk1. Erythroblasts lacking Cdk2 and Cdk4 displayed a lengthened G1 phase due to impaired phosphorylation of the retinoblastoma protein. Deletion of the retinoblastoma protein rescued the increased size displayed by erythrocytes lacking Cdk2 and Cdk4, indicating that the retinoblastoma/Cdk2/Cdk4 pathway regulates erythrocyte size. The recovery of platelet counts following a 5-fluorouracil challenge was delayed in Cdk2(fl/fl)Cdk4(-/-)vavCre mice revealing a critical role for Cdk2 and Cdk4 in stress hematopoiesis. Our data indicate that Cdk2 and Cdk4 play important overlapping roles in homeostatic and stress hematopoiesis, which need to be considered when using broad-spectrum cyclin-dependent kinase inhibitors for cancer therapy.
Subject(s)
Blood Platelets/metabolism , Cyclin-Dependent Kinase 2/genetics , Erythrocytes/cytology , Hematopoiesis/genetics , Stress, Physiological , Animals , Cell Size , Cyclin-Dependent Kinase 2/deficiency , Cyclin-Dependent Kinase 4/deficiency , Cyclin-Dependent Kinase 4/genetics , Female , Gene Deletion , Hematocrit , Hematopoietic Stem Cells/metabolism , Immunophenotyping , Male , Mice , Mice, Knockout , Phenotype , Ploidies , Retinoblastoma Protein/geneticsABSTRACT
Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. A common bottleneck across mRNA-based therapeutic approaches is the rapid design of mRNA sequences that are translationally efficient, long-lived and non-immunogenic. Currently, an accessible software tool to aid in the design of such high-quality mRNA is lacking. Here, we present mRNAid, an open-source platform for therapeutic mRNA optimization, design and visualization that offers a variety of optimization strategies for sequence and structural features, allowing one to customize desired properties into their mRNA sequence. We experimentally demonstrate that transcripts optimized by mRNAid have characteristics comparable with commercially available sequences. To encompass additional aspects of mRNA design, we experimentally show that incorporation of certain uridine analogs and untranslated regions can further enhance stability, boost protein output and mitigate undesired immunogenicity effects. Finally, this study provides a roadmap for rational design of therapeutic mRNA transcripts.
ABSTRACT
During neurogenesis, cell cycle regulators play a pivotal role in ensuring proper proliferation, cell cycle exit, and differentiation of neural precursors. However, the precise role of cyclin-dependent kinases (Cdks) in these processes is not well understood. We generated Cdk2 and Cdk4 double knockout (DKO) mice and found a striking ablation of the intermediate zone and cortical plate in mouse embryonic brain. When neural stem cells (NSCs) were isolated and analyzed, DKO NSCs proliferated comparable to wild type as Cdk1 now binds to cyclin D1 and E1 and assumes the role vacated by the loss of Cdk2 and Cdk4 in phosphorylating Rb. Although compensation was sufficient for the maintenance of self-renewal and multilineage potential, DKO NSCs displayed an altered cell cycle profile and were more prone to neuronal differentiation. This was manifested in vivo as a marked reduction in S-phase length and an increased tendency for neurogenic divisions that prevented proper expansion of the basal progenitor pool. Our data thus demonstrate the induction of neurogenic divisions in the absence of critical mediators of G1/S transition-Cdk2 and Cdk4, and highlight their evolutionary importance in the determination of cortical thickness.
Subject(s)
Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase 4/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Proliferation , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin E/genetics , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 4/genetics , Mice , Mice, Knockout , Oncogene Proteins/genetics , Oncogene Proteins/metabolismABSTRACT
Successful completion of the cell cycle relies on the precise activation and inactivation of cyclin-dependent kinases (Cdks) whose activity is mainly regulated by binding to cyclins. Recently, a new family of Cdk regulators termed Speedy/RINGO has been discovered, which can bind and activate Cdks but shares no apparent amino acid sequence homology with cyclins. All Speedy proteins share a conserved domain of approximately 140 amino acids called "Speedy Box", which is essential for Cdk binding. Speedy/RINGO proteins display an important role in oocyte maturation in Xenopus. Interestingly, a common feature of all Speedy genes is their predominant expression in testis suggesting that meiotic functions may be the most important physiological feature of Speedy genes. Speedy homologs have been reported in mammals and can be traced back to the most primitive clade of chordates (Ciona intestinalis). Here, we investigated the evolution of the Speedy genes and have identified a number of new Speedy/RINGO proteins. Through extensive analysis of numerous species, we discovered diverse evolutionary histories: the number of Speedy genes varies considerably among species, with evidence of substantial gains and losses. Despite the interspecies variation, Speedy is conserved among most species examined. Our results provide a complete picture of the Speedy gene family and its evolution.
Subject(s)
Cell Cycle Proteins/genetics , Cyclin-Dependent Kinases/metabolism , Evolution, Molecular , Amino Acid Sequence , Animals , Binding Sites , Biological Evolution , Cell Cycle/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Chickens/genetics , Chickens/metabolism , Cyclin-Dependent Kinases/chemistry , Humans , Mice , Molecular Sequence Data , Pan troglodytes/genetics , Pan troglodytes/metabolism , Phylogeny , Protein Binding , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Sharks/genetics , Sharks/metabolism , Vertebrates/genetics , Vertebrates/metabolism , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus laevis/genetics , Xenopus laevis/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
Biodegraders are targeted protein degradation constructs composed of mini-proteins/peptides linked to E3 ligase receptors. We gained deeper insights into their utility by studying Con1-SPOP, a biodegrader against proliferating cell nuclear antigen (PCNA), an oncology target. Con1-SPOP proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut) as it had more potent anti-proliferative effects and uniquely induced DNA damage, cell apoptosis, and necrosis. Proteomics showed that PCNA degradation gave impaired mitotic division and mitochondria dysfunction, effects not seen with the stoichiometric inhibitor. We further showed that doxycycline-induced Con1-SPOP achieved complete tumor growth inhibition in vivo. Intracellular delivery of mRNA encoding Con1-SPOP via lipid nanoparticles (LNPs) depleted endogenous PCNA within hours of application with nanomolar potency. Our results demonstrate the utility of biodegraders as biological tools and highlight target degradation as a more efficacious approach versus stoichiometric inhibition. Once in vivo delivery is optimized, biodegraders may be leveraged as an exciting therapeutic modality.
Subject(s)
Liposomes , Ubiquitin-Protein Ligases , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Ubiquitin-Protein Ligases/metabolism , ApoptosisABSTRACT
Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNγ) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNγ response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models-with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNγ sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.
Subject(s)
Interferon-gamma , Neoplasms , Animals , Cytokines/metabolism , Disease Models, Animal , Humans , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Intracellular Space/metabolism , Male , Mice , Protein Binding , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Mutations to RAS proteins (H-, N-, and K-RAS) are among the most common oncogenic drivers, and tumors harboring these lesions are some of the most difficult to treat. Although covalent small molecules against KRASG12C have shown promising efficacy against lung cancers, traditional barriers remain for drugging the more prevalent KRASG12D and KRASG12V mutants. Targeted degradation has emerged as an attractive alternative approach, but for KRAS, identification of the required high-affinity ligands continues to be a challenge. Another significant hurdle is the discovery of a hybrid molecule that appends an E3 ligase-recruiting moiety in a manner that satisfies the precise geometries required for productive polyubiquitin transfer while maintaining favorable druglike properties. To gain insights into the advantages and feasibility of KRAS targeted degradation, we applied a protein-based degrader (biodegrader) approach. This workflow centers on the intracellular expression of a chimeric protein consisting of a high-affinity target-binding domain fused to an engineered E3 ligase adapter. A series of anti-RAS biodegraders spanning different RAS isoform/nucleotide-state specificities and leveraging different E3 ligases provided definitive evidence for RAS degradability. Further, these established that the functional consequences of KRAS degradation are context dependent. Of broader significance, using the exquisite degradation specificity that biodegraders can possess, we demonstrated how this technology can be applied to answer questions that other approaches cannot. Specifically, application of the GDP-state specific degrader uncovered the relative prevalence of the "off-state" of WT and various KRAS mutants in the cellular context. Finally, if delivery challenges can be addressed, anti-RAS biodegraders will be exciting candidates for clinical development.
ABSTRACT
Macrocyclic peptides have the potential to address intracellular protein-protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure-activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight - the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry - an approach that has yet to succeed in the clinic despite a long history of attempts - carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers - some of the most treatment-resistant human malignancies.
ABSTRACT
We investigated p53-dependent gene expression in nitric oxide (NO)-induced apoptosis of two tumor cell types. Seventy-seven putative p53-regulated genes were screened for NO-mediated expression changes. Twenty-four genes were up-regulated and three genes were down-regulated significantly by NO in human neuroblastoma cells. Genes known to be involved in apoptosis, which were up-regulated by > or = 2-fold, included FAS, CASP-1, BIK, PUMA, DR4 and the serpins maspin (SERPINB5), and plasminogen activator inhibitor-1 (PAI-1). Real-time PCR confirmed maspin and PAI-1 mRNAs exhibited the greatest NO-induced induction, which occurred in a p53-dependent manner. The substantial NO-mediated up-regulation of these serpins mRNAs correlated with large increases in their protein levels, which occurred before or coinciding with apoptosis. p53-deficient neuroblastoma cells were largely resistant to NO killing and showed much reduced maspin and PAI-1 mRNA and protein levels after NO treatment. p53 was activated by NO mainly in the nuclei of neuroblastoma cells. p53(-/-) HCT116 colon carcinoma cells were strongly resistant to NO-induced apoptosis and failed to up-regulate maspin and PAI-1 (in contrast to p53(+/+) HCT116 cells). Our results suggest that both apoptosis and induction of the two serpins by NO require the transcriptional activity of p53. Because maspin is a tumor suppressor and PAI-1 can promote senescence and regulate cell death, it will now be worth investigating whether their p53-mediated expression contributes to the NO-induced p53-dependent death of tumor cells.
Subject(s)
Apoptosis/drug effects , Neuroblastoma/genetics , Nitric Oxide/pharmacology , Plasminogen Activator Inhibitor 1/genetics , Polymerase Chain Reaction , Serpins/genetics , Tumor Suppressor Protein p53/genetics , Cell Nucleus/pathology , Down-Regulation , Gene Expression Regulation, Neoplastic , HCT116 Cells/drug effects , HCT116 Cells/pathology , Humans , Neuroblastoma/pathology , Plasmids , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Triazenes/pharmacology , Up-RegulationABSTRACT
Sox2 is known to be important for neuron formation, but the precise mechanism through which it activates a neurogenic program and how this differs from its well-established function in self-renewal of stem cells remain elusive. In this study, we identified a highly conserved cyclin-dependent kinase (Cdk) phosphorylation site on serine 39 (S39) in Sox2. In neural stem cells (NSCs), phosphorylation of S39 enhances the ability of Sox2 to negatively regulate neuronal differentiation, while loss of phosphorylation is necessary for chromatin retention of a truncated form of Sox2 generated during neurogenesis. We further demonstrated that nonphosphorylated cleaved Sox2 specifically induces the expression of proneural genes and promotes neurogenic commitment in vivo Our present study sheds light on how the level of Cdk kinase activity directly regulates Sox2 to tip the balance between self-renewal and differentiation in NSCs.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Neurogenesis , Phosphoserine/metabolism , SOXB1 Transcription Factors/metabolism , Amino Acid Sequence , Animals , Cell Differentiation , DNA/metabolism , Gene Expression Regulation , Mice , Models, Biological , Mutant Proteins/metabolism , NIH 3T3 Cells , Neural Stem Cells/metabolism , Neurogenesis/genetics , Neurons/cytology , Neurons/metabolism , Phosphorylation , Protein Binding , Protein Stability , SOXB1 Transcription Factors/chemistry , Serine Proteases/metabolismABSTRACT
p53 tumor suppressor maintains genomic stability, typically acting through cell-cycle arrest, senescence, and apoptosis. We discovered a function of p53 in preventing conflicts between transcription and replication, independent of its canonical roles. p53 deficiency sensitizes cells to Topoisomerase (Topo) II inhibitors, resulting in DNA damage arising spontaneously during replication. Topoisomerase IIα (TOP2A)-DNA complexes preferentially accumulate in isogenic p53 mutant or knockout cells, reflecting an increased recruitment of TOP2A to regulate DNA topology. We propose that p53 acts to prevent DNA topological stress originating from transcription during the S phase and, therefore, promotes normal replication fork progression. Consequently, replication fork progression is impaired in the absence of p53, which is reversed by transcription inhibition. Pharmacologic inhibition of transcription also attenuates DNA damage and decreases Topo-II-DNA complexes, restoring cell viability in p53-deficient cells. Together, our results demonstrate a function of p53 that may underlie its role in tumor suppression.
Subject(s)
DNA Replication , Genomic Instability , Transcription, Genetic , Tumor Suppressor Protein p53/metabolism , Antigens, Neoplasm/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , HCT116 Cells , Humans , Poly-ADP-Ribose Binding Proteins , Topoisomerase Inhibitors/pharmacology , Tumor Suppressor Protein p53/geneticsABSTRACT
RATIONALE: Persistent activation of nuclear factor-kappaB has been associated with the development of asthma. Glycogen synthase kinase-3beta is known to regulate the activity of nuclear factor-kappaB. OBJECTIVES: We hypothesized that inhibition of glycogen synthase kinase-3beta may have anti-inflammatory effects in allergic asthma. METHODS: BALB/c mice sensitized and challenged with ovalbumin developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and for cytokine and chemokine levels. Lung tissues were examined for cell infiltration and mucus hypersecretion, and for the expression of inflammatory biomarkers. Serum immunoglobulin E levels were determined by enzyme-linked immunosorbant assay. Airway hyperresponsiveness was monitored by direct airway resistance analysis. MEASUREMENTS AND MAIN RESULTS: Intravenous administration of 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a selective glycogen synthase kinase-3beta inhibitor, significantly inhibited ovalbumin-induced increases in total cell counts, eosinophil counts, and IL-5, IL-13, and eotaxin levels recovered in bronchoalveolar lavage fluid in a dose-dependent manner. TDZD-8 substantially reduced the serum levels of ovalbumin-specific IgE. Histologic studies showed that TDZD-8 dramatically inhibited ovalbumin-induced lung tissue eosinophilia and airway mucus production. TDZD-8 also markedly suppressed ovalbumin-induced mRNA expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, Muc5ac, and three members of the chitinase family (acidic mammalian chitinase, Ym1, and Ym2). In addition, TDZD-8 significantly reduced ovalbumin-induced airway hyperresponsiveness to inhaled methacholine. Western blot analysis of whole lung lysates revealed that TDZD-8 markedly attenuated the phosphorylation of the nuclear factor-kappaB subunit p65 from ovalbumin-challenged mice. CONCLUSIONS: Our findings suggest that inhibition of glycogen synthase kinase-3beta may provide a novel means for the treatment of allergic airway inflammation.
Subject(s)
Asthma/drug therapy , Enzyme Inhibitors/therapeutic use , Glycogen Synthase Kinase 3/antagonists & inhibitors , NF-kappa B/antagonists & inhibitors , Thiadiazoles/therapeutic use , Analysis of Variance , Animals , Asthma/pathology , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Cytokines/metabolism , Enzyme Inhibitors/pharmacology , Eosinophils/drug effects , Female , Glycogen Synthase Kinase 3 beta , Immunoglobulin E/blood , Inflammation Mediators/metabolism , Mice , Mice, Inbred BALB C , Mucus/metabolism , Ovalbumin , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Thiadiazoles/pharmacologyABSTRACT
1. An antisense oligonucleotide (ASO) is a short strand of deoxyribonucleotide analogue that hybridizes with the complementary mRNA in a sequence-specific manner via Watson-Crick base pairing. Formation of the ASO-mRNA heteroduplex either triggers RNase H activity, leading to mRNA degradation, induces translational arrest by steric hindrance of ribosomal activity, interferes with mRNA maturation by inhibiting splicing or destabilizes pre-mRNA in the nucleus, resulting in downregulation of target protein expression. 2. The ASO is not only a useful experimental tool in protein target identification and validation, but also a highly selective therapeutic strategy for diseases with dysregulated protein expression. 3. In the present review, we discuss various theoretical approaches to rational design of ASO, chemical modifications of ASO, ASO delivery systems and ASO-related toxicology. Finally, we survey ASO drugs in various current clinical studies.