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1.
Nat Immunol ; 25(6): 1073-1082, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38816615

RESUMO

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.


Assuntos
Vacinas contra a AIDS , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Proteína gp41 do Envelope de HIV , Infecções por HIV , HIV-1 , Macaca mulatta , Animais , Humanos , Proteína gp41 do Envelope de HIV/imunologia , Anticorpos Anti-HIV/imunologia , Camundongos , Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , HIV-1/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Vacinação , Anticorpos Amplamente Neutralizantes/imunologia , Linfócitos B/imunologia , Nanopartículas/química , Feminino , Regiões Determinantes de Complementaridade/imunologia , Epitopos/imunologia
2.
Cell ; 180(2): 263-277.e20, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31955845

RESUMO

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 mya. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 million years through a process analogous to Darwinian evolution of the genome.


Assuntos
Cryptococcus neoformans/genética , Metilação de DNA/genética , Metiltransferases/genética , Evolução Biológica , Cryptococcus neoformans/metabolismo , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/fisiologia , Metilases de Modificação do DNA/genética , Elementos de DNA Transponíveis/genética , Epigenômica/métodos , Evolução Molecular , Genoma/genética , Metiltransferases/metabolismo , Filogenia
4.
Immunity ; 55(11): 2149-2167.e9, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36179689

RESUMO

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.


Assuntos
Vacinas contra a AIDS , Infecções por HIV , HIV-1 , Humanos , Anticorpos Amplamente Neutralizantes , Anticorpos Anti-HIV , Produtos do Gene env do Vírus da Imunodeficiência Humana , Anticorpos Neutralizantes , Regiões Determinantes de Complementaridade/genética , Infecções por HIV/prevenção & controle
5.
Cell ; 163(3): 583-93, 2015 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-26496605

RESUMO

LINE-1 retrotransposons are fast-evolving mobile genetic entities that play roles in gene regulation, pathological conditions, and evolution. Here, we show that the primate LINE-1 5'UTR contains a primate-specific open reading frame (ORF) in the antisense orientation that we named ORF0. The gene product of this ORF localizes to promyelocytic leukemia-adjacent nuclear bodies. ORF0 is present in more than 3,000 loci across human and chimpanzee genomes and has a promoter and a conserved strong Kozak sequence that supports translation. By virtue of containing two splice donor sites, ORF0 can also form fusion proteins with proximal exons. ORF0 transcripts are readily detected in induced pluripotent stem (iPS) cells from both primate species. Capped and polyadenylated ORF0 mRNAs are present in the cytoplasm, and endogenous ORF0 peptides are identified upon proteomic analysis. Finally, ORF0 enhances LINE-1 mobility. Taken together, these results suggest a role for ORF0 in retrotransposon-mediated diversity.


Assuntos
Pan troglodytes/genética , Retroelementos , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Citoplasma/genética , Humanos , Elementos Nucleotídeos Longos e Dispersos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta , Processamento Pós-Transcricional do RNA , RNA Antissenso/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Ribossomos/metabolismo , Alinhamento de Sequência
7.
Nature ; 609(7928): 846-853, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35940205

RESUMO

Thyroid hormones are vital in metabolism, growth and development1. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR)2. In patients with Graves' disease, autoantibodies that activate the TSHR pathologically increase thyroid hormone activity3. How autoantibodies mimic thyrotropin function remains unclear. Here we determined cryo-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. Thyrotropin selects an upright orientation of the extracellular domain owing to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody from a patient with Graves' disease selects a similar upright orientation of the extracellular domain. Reorientation of the extracellular domain transduces a conformational change in the seven-transmembrane-segment domain via a conserved hinge domain, a tethered peptide agonist and a phospholipid that binds within the seven-transmembrane-segment domain. Rotation of the TSHR extracellular domain relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to other G-protein-coupled receptors with large extracellular domains.


Assuntos
Microscopia Crioeletrônica , Imunoglobulinas Estimuladoras da Glândula Tireoide , Receptores da Tireotropina , Tireotropina , Membrana Celular/metabolismo , Doença de Graves/imunologia , Doença de Graves/metabolismo , Humanos , Imunoglobulinas Estimuladoras da Glândula Tireoide/química , Imunoglobulinas Estimuladoras da Glândula Tireoide/imunologia , Imunoglobulinas Estimuladoras da Glândula Tireoide/farmacologia , Imunoglobulinas Estimuladoras da Glândula Tireoide/ultraestrutura , Fosfolipídeos/metabolismo , Domínios Proteicos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/ultraestrutura , Receptores da Tireotropina/agonistas , Receptores da Tireotropina/química , Receptores da Tireotropina/imunologia , Receptores da Tireotropina/ultraestrutura , Rotação , Tireotropina/química , Tireotropina/metabolismo , Tireotropina/farmacologia
8.
Proc Natl Acad Sci U S A ; 120(50): e2314429120, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38055739

RESUMO

We detected ENU-induced alleles of Mfsd1 (encoding the major facilitator superfamily domain containing 1 protein) that caused lymphopenia, splenomegaly, progressive liver pathology, and extramedullary hematopoiesis (EMH). MFSD1 is a lysosomal membrane-bound solute carrier protein with no previously described function in immunity. By proteomic analysis, we identified association between MFSD1 and both GLMP (glycosylated lysosomal membrane protein) and GIMAP5 (GTPase of immunity-associated protein 5). Germline knockout alleles of Mfsd1, Glmp, and Gimap5 each caused lymphopenia, liver pathology, EMH, and lipid deposition in the bone marrow and liver. We found that the interactions of MFSD1 and GLMP with GIMAP5 are essential to maintain normal GIMAP5 expression, which in turn is critical to support lymphocyte development and liver homeostasis that suppresses EMH. These findings identify the protein complex MFSD1-GLMP-GIMAP5 operating in hematopoietic and extrahematopoietic tissues to regulate immunity and liver homeostasis.


Assuntos
Proteínas de Ligação ao GTP , Linfopenia , Humanos , Proteínas de Ligação ao GTP/metabolismo , Proteômica , Fígado/metabolismo , Linfócitos/metabolismo , Linfopenia/genética , Homeostase
9.
PLoS Genet ; 19(4): e1010710, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37068109

RESUMO

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in ß-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS ß-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS ß-cells. Consistent with reduced ER chaperones levels, PWS INS-1 ß-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS ß-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic ß-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and ß-cell secretory pathway function.


Assuntos
Síndrome de Prader-Willi , Camundongos , Animais , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/metabolismo , Secreção de Insulina/genética , Chaperona BiP do Retículo Endoplasmático , Regulação para Baixo , Proteômica , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Insulina/genética , Insulina/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo
10.
Nat Chem Biol ; 19(3): 275-283, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36175661

RESUMO

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.


Assuntos
COVID-19 , Humanos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Ligação Proteica , Peptidil Dipeptidase A/metabolismo
11.
Mol Psychiatry ; 28(4): 1813-1826, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36127429

RESUMO

Mitochondrial DNA variants have previously associated with disease, but the underlying mechanisms have been largely elusive. Here, we report that mitochondrial SNP rs2853499 associated with Alzheimer's disease (AD), neuroimaging, and transcriptomics. We mapped rs2853499 to a novel mitochondrial small open reading frame called SHMOOSE with microprotein encoding potential. Indeed, we detected two unique SHMOOSE-derived peptide fragments in mitochondria by using mass spectrometry-the first unique mass spectrometry-based detection of a mitochondrial-encoded microprotein to date. Furthermore, cerebrospinal fluid (CSF) SHMOOSE levels in humans correlated with age, CSF tau, and brain white matter volume. We followed up on these genetic and biochemical findings by carrying out a series of functional experiments. SHMOOSE acted on the brain following intracerebroventricular administration, differentiated mitochondrial gene expression in multiple models, localized to mitochondria, bound the inner mitochondrial membrane protein mitofilin, and boosted mitochondrial oxygen consumption. Altogether, SHMOOSE has vast implications for the fields of neurobiology, Alzheimer's disease, and microproteins.


Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , DNA Mitocondrial/genética , Biomarcadores/líquido cefalorraquidiano , Micropeptídeos
12.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33468658

RESUMO

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.


Assuntos
Diazometano/metabolismo , Histonas/genética , Lisina/metabolismo , Fases de Leitura Aberta , Peptídeos/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Bovinos , Cromatina/química , Cromatina/metabolismo , Diazometano/análogos & derivados , Regulação da Expressão Gênica , Loci Gênicos , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Células K562 , Lisina/análogos & derivados , Camundongos , Pan troglodytes , Peptídeos/metabolismo , Ligação Proteica/efeitos da radiação , Mapeamento de Interação de Proteínas , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos da radiação , Transgenes , Raios Ultravioleta
13.
J Proteome Res ; 22(12): 3742-3753, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37939376

RESUMO

The ß-coronavirus SARS-CoV-2 causes severe acute respiratory syndrome (COVID-19) in humans. It enters and infects epithelial airway cells upon binding of the receptor binding domain (RBD) of the virus entry protein spike to the host receptor protein Angiotensin Converting Enzyme 2 (ACE2). Here, we used coimmunoprecipitation coupled with bottom-up mass spectrometry to identify host proteins that engaged with the spike protein in human bronchial epithelial cells (16HBEo-). We found that the spike protein bound to extracellular laminin and thrombospondin and endoplasmatic reticulum (ER)-resident DJB11 and FBX2 proteins. The ER-resident proteins UGGT1, CALX, HSP7A, and GRP78/BiP bound preferentially to the original Wuhan D614 over the mutated G614 spike protein in the more rapidly spreading Alpha SARS-CoV-2 strain. The increase in protein binding to the D614 spike might be explained by higher accessibility of cryptic sites in "RDB open" and "S2 only" D614 spike protein conformations and may enable SARS-CoV-2 to infect additional, ACE2-negative cell types. Moreover, a novel proteome-based cell type set enrichment analysis (pCtSEA) found that host factors like laminin might render additional cell types such as macrophages and epithelial cells in the nephron permissive to SARS-CoV-2 infection.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Laminina , Ligação Proteica , Proteínas Virais/metabolismo , Tropismo
14.
Nucleic Acids Res ; 49(7): 3603-3616, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33341895

RESUMO

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Arginina/metabolismo , RNA de Transferência de Glutamina/metabolismo , Ribossomos/metabolismo , Animais , Fibroblastos , Células HEK293 , Humanos , Camundongos
15.
Proc Natl Acad Sci U S A ; 117(45): 28014-28025, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33093196

RESUMO

The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the "glycan shield," is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.


Assuntos
HIV-1/imunologia , Polissacarídeos/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos , Simulação por Computador , Microscopia Crioeletrônica/métodos , Epitopos/química , Glicosilação , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Soropositividade para HIV , HIV-1/metabolismo , Humanos , Evasão da Resposta Imune/imunologia , Espectrometria de Massas/métodos , Modelos Moleculares , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
16.
J Proteome Res ; 21(4): 1017-1028, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35271278

RESUMO

During tumorigenesis, DNA mutations in protein coding sequences can alter amino acid sequences which can change the structures of proteins. While the 3D structure of mutated proteins has been studied with atomic resolution, the precise impact of somatic mutations on the 3D proteome during malignant transformation remains unknown because methods to reveal in vivo protein structures in high throughput are limited. Here, we measured the accessibility of the lysine ε-amine for chemical modification across proteomes using covalent protein painting (CPP) to indirectly determine alterations in the 3D proteome. CPP is a novel, high-throughput quantitative mass spectrometric method that surveyed a total of 8052 lysine sites across the 60 cell lines of the well-studied anticancer cell line panel (NCI60). Overall, 5.2 structural alterations differentiated any cancer cell line from the other 59. Structural aberrations in 98 effector proteins correlated with the selected presence of 90 commonly mutated proteins in the NCI60 cell line panel, suggesting that different tumor genotypes reshape a limited set of effector proteins. We searched our dataset for druggable conformational aberrations and identified 49 changes in the cancer conformational landscape that correlated with the growth inhibition profiles of 300 drug candidates out of 50,000 small molecules. We found that alterations in heat shock proteins are key predictors of anticancer drug efficacy, which implies that the proteostasis network may have a general but hitherto unrecognized role in maintaining malignancy. Individual lysine sites may serve as biomarkers to guide drug selection or may be directly targeted for anticancer drug development.


Assuntos
Neoplasias , Carcinogênese/genética , Humanos , Espectrometria de Massas , Neoplasias/genética , Proteoma/química , Proteoma/genética , Proteostase
17.
Mol Psychiatry ; 26(12): 7560-7580, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34433918

RESUMO

Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Encéfalo , Deleção Cromossômica , Cromossomos Humanos Par 16/genética , Variações do Número de Cópias de DNA/genética , Humanos , Neurogênese/genética , Organoides , Proteômica
18.
Mol Psychiatry ; 26(11): 7047-7068, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33888873

RESUMO

Early-onset epileptic encephalopathies are severe disorders often associated with specific genetic mutations. In this context, the CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by early-onset seizures, intellectual delay, and motor dysfunction. Although crucial for proper brain development, the precise targets of CDKL5 and its relation to patients' symptoms are still unknown. Here, induced pluripotent stem cells derived from individuals deficient in CDKL5 protein were used to generate neural cells. Proteomic and phosphoproteomic approaches revealed disruption of several pathways, including microtubule-based processes and cytoskeleton organization. While CDD-derived neural progenitor cells have proliferation defects, neurons showed morphological alterations and compromised glutamatergic synaptogenesis. Moreover, the electrical activity of CDD cortical neurons revealed hyperexcitability during development, leading to an overly synchronized network. Many parameters of this hyperactive network were rescued by lead compounds selected from a human high-throughput drug screening platform. Our results enlighten cellular, molecular, and neural network mechanisms of genetic epilepsy that could ultimately promote novel therapeutic opportunities for patients.


Assuntos
Síndromes Epilépticas , Animais , Síndromes Epilépticas/genética , Humanos , Camundongos , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases , Proteômica
19.
Mol Psychiatry ; 26(7): 3586-3613, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33727673

RESUMO

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for autism spectrum disorder (ASD) and developmental delay (DD). To investigate how Cul3 mutations impact brain development, we generated a haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of embryonic, early postnatal and adult brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified defects in neuronal cytoskeleton and Rho signaling as the primary targets of Cul3 mutation during brain development.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Animais , Transtorno do Espectro Autista/genética , Proteínas Culina/genética , Citoesqueleto , Células Germinativas , Haploinsuficiência/genética , Camundongos , Neurogênese/genética , Proteômica
20.
Nucleic Acids Res ; 48(12): 6445-6457, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484512

RESUMO

The accuracy in pairing tRNAs with correct amino acids by aminoacyl-tRNA synthetases (aaRSs) dictates the fidelity of translation. To ensure fidelity, multiple aaRSs developed editing functions that remove a wrong amino acid from tRNA before it reaches the ribosome. However, no specific mechanism within an aaRS is known to handle the scenario where a cognate amino acid is mischarged onto a wrong tRNA, as exemplified by AlaRS mischarging alanine to G4:U69-containing tRNAThr. Here, we report that the mischargeable G4:U69-containing tRNAThr are strictly conserved in vertebrates and are ubiquitously and abundantly expressed in mammalian cells and tissues. Although these tRNAs are efficiently mischarged, no corresponding Thr-to-Ala mistranslation is detectable. Mistranslation is prevented by a robust proofreading activity of ThrRS towards Ala-tRNAThr. Therefore, while wrong amino acids are corrected within an aaRS, a wrong tRNA is handled in trans by an aaRS cognate to the mischarged tRNA species. Interestingly, although Ala-tRNAThr mischarging is not known to occur in bacteria, Escherichia coli ThrRS also possesses robust cross-editing ability. We propose that the cross-editing activity of ThrRS is evolutionarily conserved and that this intrinsic activity allows G4:U69-containing tRNAThr to emerge and be preserved in vertebrates to have alternative functions without compromising translational fidelity.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Edição de RNA , RNA de Transferência/metabolismo , Alanina/genética , Animais , Evolução Molecular , Células HEK293 , Humanos , RNA de Transferência/genética , Treonina/genética , Vertebrados/genética
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