RESUMO
Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit general translation initiation while selectively translating essential stress regulators. Unexpectedly, in plants, pattern-triggered immunity (PTI) and response to other environmental stresses occur independently of the GCN2/eIF2α pathway. Here, we show that while PTI induces mRNA decapping to inhibit general translation, defense mRNAs with a purine-rich element ("R-motif") are selectively translated using R-motif as an internal ribosome entry site (IRES). R-motif-dependent translation is executed by poly(A)-binding proteins (PABPs) through preferential association with the PTI-activating eIFiso4G over the repressive eIF4G. Phosphorylation by PTI regulators mitogen-activated protein kinase 3 and 6 (MPK3/6) inhibits eIF4G's activity while enhancing PABP binding to the R-motif and promoting eIFiso4G-mediated defense mRNA translation, establishing a link between PTI signaling and protein synthesis. Given its prevalence in both plants and animals, the PABP/R-motif translation initiation module may have a broader role in reprogramming the stress translatome.
Assuntos
Fator de Iniciação Eucariótico 4G , Proteínas de Ligação a Poli(A) , Animais , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Biossíntese de Proteínas , Purinas , RNA Mensageiro/metabolismoRESUMO
To secure phosphorus (P) from soil, most land plants use a direct phosphate uptake pathway via root hairs and epidermis and an indirect phosphate uptake pathway via mycorrhizal symbiosis. The interaction between these two pathways is unclear. Here, we mapped a network between transcription factors and mycorrhizal symbiosis-related genes using Y1H. Intriguingly, this gene regulatory network is governed by the conserved P-sensing pathway, centered on phosphate starvation response (PHR) transcription factors. PHRs are required for mycorrhizal symbiosis and regulate symbiosis-related genes via the P1BS motif. SPX-domain proteins suppress OsPHR2-mediated induction of symbiosis-related genes and inhibit mycorrhizal infection. In contrast, plants overexpressing OsPHR2 show improved mycorrhizal infection and are partially resistant to P-mediated inhibition of symbiosis. Functional analyses of network nodes revealed co-regulation of hormonal signaling and mycorrhizal symbiosis. This network deciphers extensive regulation of mycorrhizal symbiosis by endogenous and exogenous signals and highlights co-option of the P-sensing pathway for mycorrhizal symbiosis.
Assuntos
Redes Reguladoras de Genes , Micorrizas/genética , Micorrizas/fisiologia , Fosfatos/deficiência , Simbiose/genética , Simbiose/fisiologia , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Mutação/genética , Oryza/genética , Oryza/microbiologia , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.
Assuntos
COVID-19/imunologia , Aptidão Genética , Evasão da Resposta Imune , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/virologia , Humanos , Mutação , Filogenia , SARS-CoV-2/química , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , VirulênciaRESUMO
Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human ß-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human ß-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Miosinas/metabolismo , Neoplasias/tratamento farmacológico , Doenças do Sistema Nervoso/tratamento farmacológico , Infecções por Protozoários/tratamento farmacológico , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Fenômenos Biomecânicos , Cryptosporidium/efeitos dos fármacos , Cryptosporidium/enzimologia , Inibidores Enzimáticos/química , Expressão Gênica , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Família Multigênica , Mutação , Miosinas/antagonistas & inibidores , Miosinas/classificação , Miosinas/genética , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/patologia , Plasmodium/efeitos dos fármacos , Plasmodium/enzimologia , Infecções por Protozoários/enzimologia , Infecções por Protozoários/genética , Infecções por Protozoários/patologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologiaRESUMO
During autophagy, vesicle dynamics and cargo recruitment are driven by numerous adaptors and receptors that become tethered to the phagophore through interactions with lipidated ATG8/LC3 decorating the expanding membrane. Most currently described ATG8-binding proteins exploit a well-defined ATG8-interacting motif (AIM, or LC3-interacting region [LIR]) that contacts a hydrophobic patch on ATG8 known as the LIR/AIM docking site (LDS). Here we describe a new class of ATG8 interactors that exploit ubiquitin-interacting motif (UIM)-like sequences for high-affinity binding to an alternative ATG8 interaction site. Assays with candidate UIM-containing proteins together with unbiased screens identified a large collection of UIM-based ATG8 interactors in plants, yeast, and humans. Analysis of a subset also harboring ubiquitin regulatory X (UBX) domains revealed a role for UIM-directed autophagy in clearing non-functional CDC48/p97 complexes, including some impaired in human disease. With this new class of adaptors and receptors, we greatly extend the reach of selective autophagy and identify new factors regulating autophagic vesicle dynamics.
Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Motivos de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Sítios de Ligação , Humanos , Proteínas Associadas aos Microtúbulos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies."
Assuntos
Transportador de Glucose Tipo 1/fisiologia , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/fisiologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/fisiologia , Erros Inatos do Metabolismo dos Carboidratos , Clatrina/metabolismo , Citoplasma/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Proteínas Intrinsicamente Desordenadas/metabolismo , Leucina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte de Monossacarídeos/deficiência , Mutação/genética , Peptídeos , Ligação Proteica , Proteômica/métodosRESUMO
Intracellular Mg2+ (iMg2+) is bound with phosphometabolites, nucleic acids, and proteins in eukaryotes. Little is known about the intracellular compartmentalization and molecular details of Mg2+ transport into/from cellular organelles such as the endoplasmic reticulum (ER). We found that the ER is a major iMg2+ compartment refilled by a largely uncharacterized ER-localized protein, TMEM94. Conventional and AlphaFold2 predictions suggest that ERMA (TMEM94) is a multi-pass transmembrane protein with large cytosolic headpiece actuator, nucleotide, and phosphorylation domains, analogous to P-type ATPases. However, ERMA uniquely combines a P-type ATPase domain and a GMN motif for ERMg2+ uptake. Experiments reveal that a tyrosine residue is crucial for Mg2+ binding and activity in a mechanism conserved in both prokaryotic (mgtB and mgtA) and eukaryotic Mg2+ ATPases. Cardiac dysfunction by haploinsufficiency, abnormal Ca2+ cycling in mouse Erma+/- cardiomyocytes, and ERMA mRNA silencing in human iPSC-cardiomyocytes collectively define ERMA as an essential component of ERMg2+ uptake in eukaryotes.
Assuntos
Adenosina Trifosfatases , ATPases do Tipo-P , Animais , Camundongos , Humanos , Adenosina Trifosfatases/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Transporte Biológico , ATPases do Tipo-P/metabolismo , Cálcio/metabolismo , ATPases Transportadoras de Cálcio do Retículo SarcoplasmáticoRESUMO
Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function.
Assuntos
RNA , Fatores de Transcrição , Fatores de Transcrição/metabolismo , RNA/metabolismo , Sítios de Ligação , Ligação Proteica , DNA/genéticaRESUMO
Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVß5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/ß5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98 Å RMSD irisin/αVß5 complex docking model. Irisin binds very tightly to an alternative interface on αVß5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.
Assuntos
Comunicação Celular , Fibronectinas , Humanos , Fibronectinas/metabolismo , Transdução de SinaisRESUMO
Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs), which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar of a few scaffold proteins that make up these phases, but how the partitioning of hundreds of SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, an SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14 aa sequence that acts as a condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal an unexpected molecular function for ancient poly(A)-binding proteins as regulators of biomolecular condensate proteins. These findings may inspire approaches to therapeutically target aberrant phases in disease.
Assuntos
Ataxina-2 , Doenças Neurodegenerativas , Humanos , Ataxina-2/genética , Proteína I de Ligação a Poli(A) , Doenças Neurodegenerativas/metabolismo , Condensados BiomolecularesRESUMO
Pioneer transcription factors are essential for cell fate changes by targeting closed chromatin. OCT4 is a crucial pioneer factor that can induce cell reprogramming. However, the structural basis of how pioneer factors recognize the in vivo nucleosomal DNA targets is unknown. Here, we determine the high-resolution structures of the nucleosome containing human LIN28B DNA and its complexes with the OCT4 DNA binding region. Three OCT4s bind the pre-positioned nucleosome by recognizing non-canonical DNA sequences. Two use their POUS domains while the other uses the POUS-loop-POUHD region; POUHD serves as a wedge to unwrap â¼25 base pair DNA. Our analysis of previous genomic data and determination of the ESRRB-nucleosome-OCT4 structure confirmed the generality of these structural features. Moreover, biochemical studies suggest that multiple OCT4s cooperatively open the H1-condensed nucleosome array containing the LIN28B nucleosome. Thus, our study suggests a mechanism of how OCT4 can target the nucleosome and open closed chromatin.
Assuntos
Cromatina , Nucleossomos , Fator 3 de Transcrição de Octâmero , Proteínas de Ligação a RNA , Humanos , Sequência de Bases , Reprogramação Celular , Cromatina/genética , DNA/metabolismo , Nucleossomos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismoRESUMO
DNA damage repair systems are critical for genomic integrity. However, they must be coordinated with DNA replication and cell division to ensure accurate genomic transmission. In most bacteria, this coordination is mediated by the SOS response through LexA, which triggers a halt in cell division until repair is completed. Recently, an SOS-independent damage response system was revealed in Caulobacter crescentus. This pathway is controlled by the transcription activator, DriD, but how DriD senses and signals DNA damage is unknown. To address this question, we performed biochemical, cellular, and structural studies. We show that DriD binds a specific promoter DNA site via its N-terminal HTH domain to activate transcription of genes, including the cell division inhibitor didA A structure of the C-terminal portion of DriD revealed a WYL motif domain linked to a WCX dimerization domain. Strikingly, we found that DriD binds ssDNA between the WYL and WCX domains. Comparison of apo and ssDNA-bound DriD structures reveals that ssDNA binding orders and orients the DriD domains, indicating a mechanism for ssDNA-mediated operator DNA binding activation. Biochemical and in vivo studies support the structural model. Our data thus reveal the molecular mechanism underpinning an SOS-independent DNA damage repair pathway.
Assuntos
Proteínas de Bactérias , Caulobacter crescentus , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Dano ao DNA , DNA de Cadeia Simples/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Some endocrine organs are frequent targets of autoimmune attack. Here, we addressed the origin of autoimmune disease from the viewpoint of feedback control. Endocrine tissues maintain mass through feedback loops that balance cell proliferation and removal according to hormone-driven regulatory signals. We hypothesized the existence of a dedicated mechanism that detects and removes mutant cells that missense the signal and therefore hyperproliferate and hypersecrete with potential to disrupt organismal homeostasis. In this mechanism, hypersecreting cells are preferentially eliminated by autoreactive T cells at the cost of a fragility to autoimmune disease. The "autoimmune surveillance of hypersecreting mutants" (ASHM) hypothesis predicts the presence of autoreactive T cells in healthy individuals and the nature of self-antigens as peptides from hormone secretion pathway. It explains why some tissues get prevalent autoimmune disease, whereas others do not and instead show prevalent mutant-expansion disease (e.g., hyperparathyroidism). The ASHM hypothesis is testable, and we discuss experimental follow-up.
Assuntos
Doenças Autoimunes/imunologia , Diabetes Mellitus Tipo 1/imunologia , Glândulas Endócrinas/imunologia , Sistema Endócrino/imunologia , Vigilância Imunológica/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Glândulas Endócrinas/citologia , Glândulas Endócrinas/metabolismo , Sistema Endócrino/citologia , Sistema Endócrino/metabolismo , Feminino , Humanos , Vigilância Imunológica/genética , Masculino , Mutação , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2) is the positive-sense RNA virus that causes coronavirus disease 2019 (COVID-19). The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form RNA structures, yet as much as 97% of its 30 kilobases have not been structurally explored. Here, we apply a novel long amplicon strategy to determine the secondary structure of the SARS-CoV-2 RNA genome at single-nucleotide resolution in infected cells. Our in-depth structural analysis reveals networks of well-folded RNA structures throughout Orf1ab and reveals aspects of SARS-CoV-2 genome architecture that distinguish it from other RNA viruses. Evolutionary analysis shows that several features of the SARS-CoV-2 genomic structure are conserved across ß-coronaviruses, and we pinpoint regions of well-folded RNA structure that merit downstream functional analysis. The native, secondary structure of SARS-CoV-2 presented here is a roadmap that will facilitate focused studies on the viral life cycle, facilitate primer design, and guide the identification of RNA drug targets against COVID-19.
Assuntos
COVID-19 , Genoma Viral , Conformação de Ácido Nucleico , RNA Viral , Elementos de Resposta , SARS-CoV-2 , COVID-19/genética , COVID-19/metabolismo , Linhagem Celular Tumoral , Humanos , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMO
Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.
Assuntos
Calcineurina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Biotinilação , Centrossomo/metabolismo , Simulação por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Monoéster Fosfórico Hidrolases/química , Fosforilação , Mapas de Interação de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
Dynamic protein phosphorylation and dephosphorylation are essential regulatory mechanisms that ensure proper cellular signaling and biological functions. Deregulation of either reaction has been implicated in several human diseases. Here, we focus on the mechanisms that govern the specificity of the dephosphorylation reaction. Most cellular serine/threonine dephosphorylation is catalyzed by 13 highly conserved phosphoprotein phosphatase (PPP) catalytic subunits, which form hundreds of holoenzymes by binding to regulatory and scaffolding subunits. PPP holoenzymes recognize phosphorylation site consensus motifs and interact with short linear motifs (SLiMs) or structural elements distal to the phosphorylation site. We review recent advances in understanding the mechanisms of PPP site-specific dephosphorylation preference and substrate recruitment and highlight examples of their interplay in the regulation of cell division.
Assuntos
Fosfoproteínas Fosfatases , Humanos , Fosforilação , Fosfoproteínas Fosfatases/metabolismo , Domínio Catalítico , Holoenzimas/química , Holoenzimas/metabolismo , Especificidade por SubstratoRESUMO
Centromere protein A (CENP-A) nucleosomes containing the centromere-specific histone H3 variant CENP-A represent an epigenetic mark that specifies centromere position. The Mis18 complex is a licensing factor for new CENP-A deposition via the CENP-A chaperone, Holliday junction recognition protein (HJURP), on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP-C-like motif, and our previous study suggested that ggKNL2 directly binds to the CENP-A nucleosome to recruit HJURP/CENP-A to the centromere. However, the molecular basis for CENP-A nucleosome recognition by ggKNL2 has remained unclear. Here, we present the cryo-EM structure of the chicken CENP-A nucleosome in complex with a ggKNL2 fragment containing the CENP-C-like motif. Chicken KNL2 distinguishes between CENP-A and histone H3 in the nucleosome using the CENP-C-like motif and its downstream region. Both the C-terminal tail and the RG-loop of CENP-A are simultaneously recognized as CENP-A characteristics. The CENP-A nucleosome-ggKNL2 interaction is thus essential for KNL2 functions. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 changes its binding partner at the centromere during chicken cell cycle progression.
Assuntos
Histonas , Nucleossomos , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Microscopia Crioeletrônica , Histonas/metabolismo , Proteínas de Ligação a DNA/química , Animais , GalinhasRESUMO
Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress.
Assuntos
DNA , Quadruplex G , Humanos , Genoma Humano , Nucleotidiltransferases , Replicação do DNARESUMO
Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.
Assuntos
Epilepsia Generalizada , Glutamato-Amônia Ligase , Glutamina , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Epilepsia Generalizada/genética , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Glutamatos/metabolismo , Glutamina/genética , Glutamina/metabolismoRESUMO
Vertebrate motile cilia are classified as (9+2) or (9+0), based on the presence or absence of the central pair apparatus, respectively. Cryogenic electron microscopy analyses of (9+2) cilia have uncovered an elaborate axonemal protein composition. The extent to which these features are conserved in (9+0) cilia remains unclear. CFAP53, a key axonemal filamentous microtubule inner protein (fMIP) and a centriolar satellites component, is essential for motility of (9+0), but not (9+2) cilia. Here, we show that in (9+2) cilia, CFAP53 functions redundantly with a paralogous fMIP, MNS1. MNS1 localises to ciliary axonemes, and combined loss of both proteins in zebrafish and mice caused severe outer dynein arm loss from (9+2) cilia, significantly affecting their motility. Using immunoprecipitation, we demonstrate that, whereas MNS1 can associate with itself and CFAP53, CFAP53 is unable to self-associate. We also show that additional axonemal dynein-interacting proteins, two outer dynein arm docking (ODAD) complex members, show differential localisation between types of motile cilia. Together, our findings clarify how paralogous fMIPs, CFAP53 and MNS1, function in regulating (9+2) versus (9+0) cilia motility, and further emphasise extensive structural diversity among these organelles.