ABSTRACT
The evolutionarily conserved HIRA/Hir histone chaperone complex and ASF1a/Asf1 co-chaperone cooperate to deposit histone (H3/H4)2 tetramers on DNA for replication-independent chromatin assembly. The molecular architecture of the HIRA/Hir complex and its mode of histone deposition have remained unknown. Here, we report the cryo-EM structure of the S. cerevisiae Hir complex with Asf1/H3/H4 at 2.9-6.8 Å resolution. We find that the Hir complex forms an arc-shaped dimer with a Hir1/Hir2/Hir3/Hpc2 stoichiometry of 2/4/2/4. The core of the complex containing two Hir1/Hir2/Hir2 trimers and N-terminal segments of Hir3 forms a central cavity containing two copies of Hpc2, with one engaged by Asf1/H3/H4, in a suitable position to accommodate a histone (H3/H4)2 tetramer, while the C-terminal segments of Hir3 harbor nucleic acid binding activity to wrap DNA around the Hpc2-assisted histone tetramer. The structure suggests a model for how the Hir/Asf1 complex promotes the formation of histone tetramers for their subsequent deposition onto DNA.
Subject(s)
Cell Cycle Proteins , Cryoelectron Microscopy , Histone Chaperones , Histones , Protein Binding , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Histones/metabolism , Histones/chemistry , Histones/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/ultrastructure , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Histone Chaperones/metabolism , Histone Chaperones/chemistry , Histone Chaperones/genetics , Models, Molecular , Molecular Chaperones/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Protein Multimerization , Binding Sites , Transcription Factors/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Protein Interaction Domains and MotifsABSTRACT
Several rRNA-modifying enzymes install rRNA modifications while participating in ribosome assembly. Here, we show that 18S rRNA methyltransferase DIMT1 is essential for acute myeloid leukemia (AML) proliferation through a noncatalytic function. We reveal that targeting a positively charged cleft of DIMT1, remote from the catalytic site, weakens the binding of DIMT1 to rRNA and mislocalizes DIMT1 to the nucleoplasm, in contrast to the primarily nucleolar localization of wild-type DIMT1. Mechanistically, rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation, which explains the distinct nucleoplasm localization of the rRNA binding-deficient DIMT1. Re-expression of wild-type or a catalytically inactive mutant E85A, but not the rRNA binding-deficient DIMT1, supports AML cell proliferation. This study provides a new strategy to target DIMT1-regulated AML proliferation via targeting this essential noncatalytic region.
Subject(s)
Leukemia, Myeloid, Acute , Methyltransferases , Humans , Cell Nucleolus/metabolism , Cell Nucleus/metabolism , Leukemia, Myeloid, Acute/genetics , Methyltransferases/metabolism , RNA Processing, Post-Transcriptional , RNA, Ribosomal, 18S/metabolismABSTRACT
Protein quality control systems are crucial for cellular function and organismal health. At present, most known protein quality control systems are multicomponent machineries that operate via ATP-regulated interactions with non-native proteins to prevent aggregation and promote folding1, and few systems that can broadly enable protein folding by a different mechanism have been identified. Moreover, proteins that contain the extensively charged poly-Asp/Glu (polyD/E) region are common in eukaryotic proteomes2, but their biochemical activities remain undefined. Here we show that DAXX, a polyD/E protein that has been implicated in diverse cellular processes3-10, possesses several protein-folding activities. DAXX prevents aggregation, solubilizes pre-existing aggregates and unfolds misfolded species of model substrates and neurodegeneration-associated proteins. Notably, DAXX effectively prevents and reverses aggregation of its in vivo-validated client proteins, the tumour suppressor p53 and its principal antagonist MDM2. DAXX can also restore native conformation and function to tumour-associated, aggregation-prone p53 mutants, reducing their oncogenic properties. These DAXX activities are ATP-independent and instead rely on the polyD/E region. Other polyD/E proteins, including ANP32A and SET, can also function as stand-alone, ATP-independent molecular chaperones, disaggregases and unfoldases. Thus, polyD/E proteins probably constitute a multifunctional protein quality control system that operates via a distinctive mechanism.
Subject(s)
Co-Repressor Proteins/metabolism , Molecular Chaperones/metabolism , Protein Folding , Animals , Cell Line , Cells/metabolism , Evolution, Molecular , Humans , Models, Molecular , Mutation , Protein Aggregates , Protein Aggregation, Pathological/prevention & control , Protein Conformation , Protein Domains , Protein Unfolding , Proteostasis Deficiencies/prevention & control , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
N-terminal acetylation (NTA) is one of the most widespread protein modifications, which occurs on most eukaryotic proteins, but is significantly less common on bacterial and archaea proteins. This modification is carried out by a family of enzymes called N-terminal acetyltransferases (NATs). To date, 12 NATs have been identified, harboring different composition, substrate specificity, and in some cases, modes of regulation. Recent structural and biochemical analysis of NAT proteins allows for a comparison of their molecular mechanisms and modes of regulation, which are described here. Although sharing an evolutionarily conserved fold and related catalytic mechanism, each catalytic subunit uses unique elements to mediate substrate-specific activity, and use NAT-type specific auxiliary and regulatory subunits, for their cellular functions.
Subject(s)
Acetyltransferases/chemistry , Acetylation , Protein Conformation , Protein Processing, Post-TranslationalABSTRACT
The HIRA histone chaperone complex is comprised of four protein subunits: HIRA, UBN1, CABIN1, and transiently associated ASF1a. All four subunits have been demonstrated to play a role in deposition of the histone variant H3.3 onto areas of actively transcribed euchromatin in cells. The mechanism by which these subunits function together to drive histone deposition has remained poorly understood. Here we present biochemical and biophysical data supporting a model whereby ASF1a delivers histone H3.3/H4 dimers to the HIRA complex, H3.3/H4 tetramerization drives the association of two HIRA/UBN1 complexes, and the affinity of the histones for DNA drives release of ASF1a and subsequent histone deposition. These findings have implications for understanding how other histone chaperone complexes may mediate histone deposition.
ABSTRACT
ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within an intrinsically disordered region and includes at least one dynamically phosphorylated residue. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the percent spliced in (PSI) of exon 14 is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types. This prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.
Subject(s)
ATP Citrate (pro-S)-Lyase , Alternative Splicing , Neoplasms , Humans , Animals , Mice , ATP Citrate (pro-S)-Lyase/metabolism , ATP Citrate (pro-S)-Lyase/genetics , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Phenotype , Exons , AcetylationABSTRACT
Human glucose-6-phosphate dehydrogenase (G6PD) is the main cellular source of NADPH, and thus plays a key role in maintaining reduced glutathione to protect cells from oxidative stress disorders such as hemolytic anemia. G6PD is a multimeric enzyme that uses the cofactors ß-D-glucose 6-phosphate (G6P) and "catalytic" NADP+ (NADP+c), as well as a "structural" NADP+ (NADP+s) located â¼25 Å from the active site, to generate NADPH. While X-ray crystallographic and biochemical studies have revealed a role for NADP+s in maintaining the catalytic activity by stabilizing the multimeric G6PD conformation, other potential roles for NADP+s have not been evaluated. Here, we determined the high resolution cryo-electron microscopy structures of human wild-type G6PD in the absence of bound ligands and a catalytic G6PD-D200N mutant bound to NADP+c and NADP+s in the absence or presence of G6P. A comparison of these structures, together with previously reported structures, reveals that the unliganded human G6PD forms a mixture of dimers and tetramers with similar overall folds, and binding of NADP+s induces a structural ordering of a C-terminal extension region and allosterically regulates G6P binding and catalysis. These studies have implications for understanding G6PD deficiencies and for therapy of G6PD-mediated disorders.
Subject(s)
Glucosephosphate Dehydrogenase , NADP , Catalytic Domain/genetics , Cryoelectron Microscopy , Glucosephosphate Dehydrogenase/chemistry , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase Deficiency/drug therapy , Glucosephosphate Dehydrogenase Deficiency/enzymology , Humans , Mutation , NADP/chemistry , Protein MultimerizationABSTRACT
Copalyl diphosphate synthase from Penicillium fellutanum (PfCPS) is an assembly-line terpene synthase that contains both prenyltransferase and class II cyclase activities. The prenyltransferase catalyzes processive chain elongation reactions using dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate to yield geranylgeranyl diphosphate, which is then utilized as a substrate by the class II cyclase domain to generate copalyl diphosphate. Here, we report the 2.81 Å-resolution cryo-EM structure of the hexameric prenyltransferase of full-length PfCPS, which is surrounded by randomly splayed-out class II cyclase domains connected by disordered polypeptide linkers. The hexamer can be described as a trimer of dimers; surprisingly, one of the three dimer-dimer interfaces is separated to yield an open hexamer conformation, thus breaking the D3 symmetry typically observed in crystal structures of other prenyltransferase hexamers such as wild-type human GGPP synthase (hGGPPS). Interestingly, however, an open hexamer conformation was previously observed in the crystal structure of D188Y hGGPPS, apparently facilitated by hexamer-hexamer packing in the crystal lattice. The cryo-EM structure of the PfCPS prenyltransferase hexamer is the first to reveal that an open conformation can be achieved even in the absence of a point mutation or interaction with another hexamer. Even though PfCPS octamers are not detected, we suggest that the open hexamer conformation represents an intermediate in the hexamer-octamer equilibrium for those prenyltransferases that do exhibit oligomeric heterogeneity.
Subject(s)
Alkyl and Aryl Transferases , Dimethylallyltranstransferase , Penicillium , Humans , Dimethylallyltranstransferase/genetics , Penicillium/genetics , Plant Proteins/geneticsABSTRACT
In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online.
ABSTRACT
The heme-regulated inhibitor (HRI) is a heme-sensing kinase that regulates mRNA translation in erythroid cells. In heme deficiency, HRI is activated to phosphorylate eukaryotic initiation factor 2α and halt production of globins, thus avoiding accumulation of heme-free globin chains. HRI is inhibited by heme via binding to one or two heme-binding domains within the HRI N-terminal and kinase domains. HRI has recently been found to inhibit fetal hemoglobin (HbF) production in adult erythroid cells. Depletion of HRI increases HbF production, presenting a therapeutically exploitable target for the treatment of patients with sickle cell disease or thalassemia, which benefit from elevated HbF levels. HRI is known to be an oligomeric enzyme that is activated through autophosphorylation, although the exact nature of the HRI oligomer, its relation to autophosphorylation, and its mode of heme regulation remain unclear. Here, we employ biochemical and biophysical studies to demonstrate that HRI forms a dimeric species that is not dependent on autophosphorylation, the C-terminal coiled-coil domain in HRI is essential for dimer formation, and dimer formation facilitates efficient autophosphorylation and activation of HRI. We also employ kinetic studies to demonstrate that the primary avenue by which heme inhibits HRI is through the heme-binding site within the kinase domain, and that this inhibition is relatively independent of binding of ATP and eukaryotic initiation factor 2α substrates. Together, these studies highlight the mode of heme inhibition and the importance of dimerization in human HRI heme-sensing activity.
Subject(s)
Heme , eIF-2 Kinase , Humans , Dimerization , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolism , Eukaryotic Initiation Factor-2/metabolism , Heme/metabolism , Kinetics , Phosphorylation , Protein BindingABSTRACT
N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases. A major member of this enzyme family, NatB, acts on much of the human proteome, including α-synuclein (αS), a synaptic protein that mediates vesicle trafficking. NatB acetylation of αS modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinson's disease. Although the molecular details of the interaction between human NatB (hNatB) and the N-terminus of αS have been resolved, whether the remainder of the protein plays a role in interacting with the enzyme is unknown. Here, we execute the first synthesis, by native chemical ligation, of a bisubstrate inhibitor of NatB consisting of coenzyme A and full-length human αS, additionally incorporating two fluorescent probes for studies of conformational dynamics. We use cryo-electron microscopy (cryo-EM) to characterize the structural features of the hNatB/inhibitor complex and show that, beyond the first few residues, αS remains disordered when in complex with hNatB. We further probe changes in the αS conformation by single molecule Förster resonance energy transfer (smFRET) to reveal that the C-terminus expands when bound to hNatB. Computational models based on the cryo-EM and smFRET data help to explain the conformational changes as well as their implications for hNatB substrate recognition and specific inhibition of the interaction with αS. Beyond the study of αS and NatB, these experiments illustrate valuable strategies for the study of challenging structural biology targets through a combination of protein semi-synthesis, cryo-EM, smFRET, and computational modeling.
Subject(s)
Parkinson Disease , alpha-Synuclein , Humans , alpha-Synuclein/chemistry , N-Terminal Acetyltransferases , Cryoelectron MicroscopyABSTRACT
The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.
Subject(s)
Cell Lineage , Heterocyclic Compounds, 4 or More Rings/pharmacology , Heterocyclic Compounds, 4 or More Rings/therapeutic use , Histone Acetyltransferases/antagonists & inhibitors , Neoplasms/drug therapy , Neoplasms/pathology , p300-CBP Transcription Factors/antagonists & inhibitors , Acetyl Coenzyme A/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Binding, Competitive , Biocatalysis/drug effects , Catalytic Domain/drug effects , Cell Line, Tumor , Cell Lineage/drug effects , Cell Proliferation/drug effects , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Gene Expression Regulation, Neoplastic/drug effects , Hematologic Neoplasms/drug therapy , Hematologic Neoplasms/enzymology , Hematologic Neoplasms/pathology , Heterocyclic Compounds, 4 or More Rings/chemistry , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/metabolism , Humans , Male , Mice , Mice, SCID , Models, Molecular , Neoplasms/enzymology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/enzymology , Prostatic Neoplasms, Castration-Resistant/pathology , Protein Conformation , Receptors, Androgen/metabolism , Xenograft Model Antitumor Assays , p300-CBP Transcription Factors/chemistry , p300-CBP Transcription Factors/metabolismABSTRACT
Huntington's disease (HD) is a neurodegenerative disorder caused by a poly-CAG expansion in the first exon of the HTT gene, resulting in an extended poly-glutamine tract in the N-terminal domain of the Huntingtin (Htt) protein product. Proteolytic fragments of the poly-glutamine-containing N-terminal domain form intranuclear aggregates that are correlated with HD. Post-translational modification of Htt has been shown to alter its function and aggregation properties. However, the effect of N-terminal Htt acetylation has not yet been considered. Here, we developed a bacterial system to produce unmodified or N-terminally acetylated and aggregation-inducible Htt protein. We used this system together with biochemical, biophysical, and imaging studies to confirm that the Htt N-terminus is an in vitro substrate for the NatA N-terminal acetyltransferase and show that N-terminal acetylation promotes aggregation. These studies represent the first link between N-terminal acetylation and the promotion of a neurodegenerative disease and implicates NatA-mediated Htt acetylation as a new potential therapeutic target in HD.
Subject(s)
Huntingtin Protein/metabolism , Protein Aggregates , Acetylation , Humans , Huntington Disease/genetics , N-Terminal Acetyltransferase A/metabolism , Peptides/metabolism , Protein Processing, Post-TranslationalABSTRACT
Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.
Subject(s)
Copper/metabolism , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , Molecular Chaperones/metabolism , Cell Line , Enzyme Activation , Humans , Protein BindingABSTRACT
N-alpha-acetylation is one of the most common co-translational protein modifications in humans and is essential for normal cell function. NAA10 encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. The auxiliary and regulatory subunits of the NatA complex are NAA15 and Huntington-interacting protein (HYPK), respectively. Through a genotype-first approach with exome sequencing, we identified and phenotypically characterized 30 individuals from 30 unrelated families with 17 different de novo or inherited, dominantly acting missense variants in NAA10 or NAA15. Clinical features of affected individuals include variable levels of intellectual disability, delayed speech and motor milestones and autism spectrum disorder. Additionally, some subjects present with mild craniofacial dysmorphology, congenital cardiac anomalies and seizures. One of the individuals is an 11-year-old boy with a frameshift variant in exon 7 of NAA10, who presents most notably with microphthalmia, which confirms a prior finding with a single family with Lenz microphthalmia syndrome. Biochemical analyses of variants as part of the human NatA complex, as well as enzymatic analyses with and without the HYPK regulatory subunit, help to explain some of the phenotypic differences seen among the different variants.
Subject(s)
Biomarkers , Genetic Association Studies , Genetic Predisposition to Disease , Genetic Variation , N-Terminal Acetyltransferase A/genetics , N-Terminal Acetyltransferase E/genetics , Phenotype , Adolescent , Adult , Alleles , Child , Child, Preschool , Computational Biology/methods , Enzyme Activation , Enzyme Stability , Facies , Female , Genetic Loci , Genetic Testing , Genotype , Humans , Infant , Male , Models, Molecular , Mutation , N-Terminal Acetyltransferase A/chemistry , N-Terminal Acetyltransferase A/metabolism , N-Terminal Acetyltransferase E/chemistry , N-Terminal Acetyltransferase E/metabolism , Protein Conformation , Recombinant Proteins , Structure-Activity Relationship , Young AdultABSTRACT
p300 and CREB-binding protein (CBP) are essential for a multitude of cellular processes. Dysregulation of p300/CBP histone acetyltransferase activity is linked to a broad spectrum of human diseases including cancers. A novel drug-like spirohydantoin (21) has been discovered as a selective orally bioavailable inhibitor of p300/CBP histone acetyltransferase. Lead compound 21 is more potent than the first-in-class lead A-485 in both enzymatic and cellular assays and lacks the off-target inhibition of dopamine and serotonin transporters, that was observed with A-485.
Subject(s)
CREB-Binding Protein/antagonists & inhibitors , Drug Discovery , E1A-Associated p300 Protein/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Hydantoins/pharmacology , Spiro Compounds/pharmacology , Administration, Oral , Biological Availability , CREB-Binding Protein/metabolism , Dose-Response Relationship, Drug , E1A-Associated p300 Protein/metabolism , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/metabolism , Humans , Hydantoins/administration & dosage , Hydantoins/metabolism , Molecular Structure , Spiro Compounds/administration & dosage , Spiro Compounds/metabolism , Structure-Activity RelationshipABSTRACT
Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.
Subject(s)
Autophagy , Nuclear Lamina/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Autophagy-Related Protein 8 Family , Cell Transformation, Neoplastic , Cells, Cultured , Cellular Senescence , Chromatin/chemistry , Chromatin/metabolism , Cytoplasm/metabolism , Fibroblasts , HEK293 Cells , Humans , Lamin Type B/genetics , Lamin Type B/metabolism , Lysosomes/metabolism , Mice , Microfilament Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Oncogene Protein p21(ras)/metabolism , Protein Binding , ProteolysisABSTRACT
N-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation. In this work, we developed potent and selective bisubstrate inhibitors for NAA80 and determined the crystal structure of NAA80 in complex with such an inhibitor, revealing that NAA80 adopts a fold similar to other NAT enzymes but with a more open substrate binding region. Furthermore, in contrast to most other NATs, the substrate specificity of NAA80 is mainly derived through interactions between the enzyme and the acidic amino acids at positions 2 and 3 of the actin substrate and not residues 1 and 2. A yeast model revealed that ectopic expression of NAA80 in a strain lacking NatB activity partially restored Nt acetylation of NatB substrates, including yeast actin. Thus, NAA80 holds intrinsic capacity to posttranslationally Nt acetylate NatB-type substrates in vivo. In sum, the presence of a dominant cotranslational NatB in all eukaryotes, the specific posttranslational actin methionine removal in animals, and finally, the unique structural features of NAA80 leave only the processed actins as in vivo substrates of NAA80. Together, this study reveals the molecular and cellular basis of NAA80 Nt acetylation and provides a scaffold for development of inhibitors for the regulation of cytoskeletal properties.
Subject(s)
Acetyltransferases/chemistry , Enzyme Inhibitors/chemistry , N-Terminal Acetyltransferases/chemistry , Actins/chemistry , Crystallography, X-Ray , Humans , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Structure-Activity RelationshipABSTRACT
In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and â¼3% of all cancers, and many drugs target the ATP binding site of the enzyme for its inhibition. Because B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an α-helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We assessed the binding between B-Raf mutants and MEK using pull downs and biolayer interferometry and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this α-helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf α-helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.
Subject(s)
MAP Kinase Kinase 1/chemistry , MAP Kinase Kinase 1/metabolism , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/metabolism , Allosteric Site , Amino Acid Sequence , Drug Discovery , Drug Resistance, Neoplasm , HEK293 Cells , Humans , In Vitro Techniques , Kinetics , MAP Kinase Kinase 1/genetics , MAP Kinase Signaling System , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Phosphorylation , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins B-raf/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static ElectricityABSTRACT
ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain in the mechanism of acetyl-CoA formation. Using recombinant, purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrated that the C terminus maintains ACLY tetramerization, a conserved and essential quaternary structure in vitro and likely also in vivo Furthermore, we show that the C terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA. Together, we demonstrate that ACLY forms a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA-dependent cellular processes for potential therapeutic use.