RESUMO
Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.
Assuntos
Mitocôndrias/metabolismo , Degeneração Neural/metabolismo , Mapas de Interação de Proteínas , Sinapses/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Aminoácidos/metabolismo , Biotinilação , Encéfalo/metabolismo , Encéfalo/patologia , Núcleo Celular/metabolismo , Progressão da Doença , Metabolismo Energético , Demência Frontotemporal/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Degeneração Neural/patologia , Neurônios/metabolismo , Ligação Proteica , Domínios Proteicos , Proteômica , Índice de Gravidade de Doença , Frações Subcelulares/metabolismo , Tauopatias/genética , Proteínas tau/químicaRESUMO
V2-glycan/apex broadly neutralizing antibodies (bnAbs) recognize a closed quaternary epitope of the HIV-1 envelope glycoprotein (Env). This closed structure is necessary to elicit apex antibodies and useful to guide the maturation of other bnAb classes. To compare antigens designed to maintain this conformation, we evaluated apex-specific responses in mice engrafted with a diverse repertoire of B cells expressing the HCDR3 of the apex bnAb VRC26.25. Engineered B cells affinity matured, guiding the improvement of VRC26.25 itself. We found that soluble Env (SOSIP) variants differed significantly in their ability to raise anti-apex responses. A transmembrane SOSIP (SOSIP-TM) delivered as an mRNA-lipid nanoparticle elicited more potent neutralizing responses than multimerized SOSIP proteins. Importantly, SOSIP-TM elicited neutralizing sera from B cells engineered with the predicted VRC26.25-HCDR3 progenitor, which also affinity matured. Our data show that HCDR3-edited B cells facilitate efficient in vivo comparisons of Env antigens and highlight the potential of an HCDR3-focused vaccine approach.
Assuntos
Vacinas contra a AIDS , Infecções por HIV , HIV-1 , Vacinas , Animais , Camundongos , Anticorpos Anti-HIV , Anticorpos Neutralizantes , Anticorpos Amplamente Neutralizantes , Antígenos Virais , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.
Assuntos
Ascorbato Peroxidases/química , Mapas de Interação de Proteínas , Coloração e Rotulagem/métodos , Animais , Humanos , Lisossomos/metabolismo , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides/metabolismo , Ubiquitina/metabolismoRESUMO
G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the ß2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of ß2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.
Assuntos
Ascorbato Peroxidases/química , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais , Coloração e Rotulagem/métodos , Ascorbato Peroxidases/metabolismo , Biotina/química , Proteínas de Ligação ao GTP/análise , Células HEK293 , Humanos , Oligopeptídeos/farmacologia , Engenharia de Proteínas , Receptor Tipo 1 de Angiotensina/agonistas , beta-Arrestinas/químicaRESUMO
Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.
Assuntos
Vacinas contra a AIDS , Infecções por HIV , HIV-1 , Camundongos , Humanos , Animais , Anticorpos Anti-HIV , Anticorpos Amplamente Neutralizantes , Anticorpos Neutralizantes , RNA Mensageiro/genética , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
Microhomology-mediated end joining (MMEJ) is an intrinsically mutagenic pathway of DNA double-strand break (DSB) repair essential for proliferation of homologous recombination (HR)-deficient tumors. Although targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as an MMEJ effector. We show that loss of APE2 inhibits MMEJ at deprotected telomeres and at intra-chromosomal DSBs and is epistatic with Pol Theta for MMEJ activity. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease activity, and further identify an uncharacterized domain required for its recruitment to DSBs. We conclude that this previously unappreciated role of APE2 in MMEJ contributes to the addiction of HRD cells to APE2, which could be exploited in the treatment of cancer.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Recombinação HomólogaRESUMO
The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.
Assuntos
Núcleo Celular , Proteínas de Ligação a RNA , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Humanos , Íntrons/genética , Splicing de RNA , Proteínas de Ligação a RNA/genéticaRESUMO
Autophagy deficiency in fed conditions leads to the formation of protein inclusions highlighting the contribution of this lysosomal delivery route to cellular proteostasis. Selective autophagy pathways exist that clear accumulated and aggregated ubiquitinated proteins. Receptors for this type of autophagy (aggrephagy) include p62, NBR1, TOLLIP, and OPTN, which possess LC3-interacting regions and ubiquitin-binding domains (UBDs), thus working as a bridge between LC3/GABARAP proteins and ubiquitinated substrates. However, the identity of aggrephagy substrates and the redundancy of aggrephagy and related UBD-containing receptors remains elusive. Here, we combined proximity labeling and organelle enrichment with quantitative proteomics to systematically map the autophagic degradome targeted by UBD-containing receptors under basal and proteostasis-challenging conditions in human cell lines. We identified various autophagy substrates, some of which were differentially engulfed by autophagosomal and endosomal membranes via p62 and TOLLIP, respectively. Overall, this resource will allow dissection of the proteostasis contribution of autophagy to numerous individual proteins.
Assuntos
Autofagossomos , Autofagia , Mapas de Interação de Proteínas , Proteólise , Proteostase , Ubiquitinação , Autofagossomos/genética , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Células HEK293 , Células HeLa , Humanos , ProteômicaRESUMO
The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with â¼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Corpos de Inclusão Intranuclear/genética , Cromossomo Y/genética , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , DNA Metiltransferase 3A , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células-Tronco Embrionárias/fisiologia , Endonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Corpos de Inclusão Intranuclear/metabolismo , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Enzimas Multifuncionais/genética , Família Multigênica , Estresse Oxidativo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteínas/genética , Fatores de Transcrição/genética , Cromossomo Y/metabolismoRESUMO
Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Proteína C9orf72/genética , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/patologia , Dipeptídeos/genética , Dipeptídeos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Camundongos , Proteômica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Enzimas Multifuncionais/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular , DNA/metabolismo , Dano ao DNA , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Genes BRCA1/fisiologia , Humanos , Enzimas Multifuncionais/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismoRESUMO
Diverse ribonucleoprotein complexes control mRNA processing, translation, and decay. Transcripts in these complexes localize to specific regions of the cell and can condense into non-membrane-bound structures such as stress granules. It has proven challenging to map the RNA composition of these large and dynamic structures, however. We therefore developed an RNA proximity labeling technique, APEX-seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching the spatial transcriptome, as revealed by APEX-seq, with the spatial proteome determined by APEX-mass spectrometry (APEX-MS), obtained precisely in parallel, provides new insights into the organization of translation initiation complexes on active mRNAs and unanticipated complexity in stress granule composition. Our novel technique allows a powerful and general approach to explore the spatial environment of macromolecules.
Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Enzimas Multifuncionais/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA/metabolismo , Coloração e Rotulagem , Transcriptoma , Grânulos Citoplasmáticos/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Células HEK293 , Humanos , Enzimas Multifuncionais/genética , RNA/genéticaRESUMO
BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.
Assuntos
Proteína BRCA2/genética , Sistemas CRISPR-Cas , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases Flap/genética , Genes Letais , Neoplasias/genética , Interferência de RNA , Mutações Sintéticas Letais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Morte Celular , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases , Endonucleases Flap/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Enzimas Multifuncionais , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genéticaRESUMO
Broadly neutralizing antibodies (bnAbs) to HIV delineate vaccine targets and are prophylactic and therapeutic agents. Some of the most potent bnAbs target a quaternary epitope at the apex of the surface HIV envelope (Env) trimer. Using cryo-electron microscopy, we solved the atomic structure of an apex bnAb, PGT145, in complex with Env. We showed that the long anionic HCDR3 of PGT145 penetrated between glycans at the trimer 3-fold axis, to contact peptide residues from all three Env protomers, and thus explains its highly trimer-specific nature. Somatic hypermutation in the other CDRs of PGT145 were crucially involved in stabilizing the structure of the HCDR3, similar to bovine antibodies, to aid in recognition of a cluster of conserved basic residues hypothesized to facilitate trimer disassembly during viral entry. Overall, the findings exemplify the creative solutions that the human immune system can evolve to recognize a conserved motif buried under a canopy of glycans.
Assuntos
Anticorpos Neutralizantes/química , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Sequência de Aminoácidos , Ânions/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Células HEK293 , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Modelos Moleculares , Polissacarídeos/química , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Ligação Proteica/imunologia , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Apex broadly neutralizing HIV antibodies (bnAbs) recognize glycans and protein surface close to the 3-fold axis of the envelope (Env) trimer and are among the most potent and broad Abs described. The evolution of apex bnAbs from one donor (CAP256) has been studied in detail and many Abs at different stages of maturation have been described. Using diverse engineering tools, we investigated the involvement of glycan recognition in the development of the CAP256.VRC26 Ab lineage. We found that sialic acid-bearing glycans were recognized by germline-encoded and somatically mutated residues on the Ab heavy chain. This recognition provided an "anchor" for the Abs as the core protein epitope varies, prevented complete neutralization escape, and eventually led to broadening of the response. These findings illustrate how glycan-specific maturation enables a human Ab to cope with pathogen escape mechanisms and will aid in optimization of immunization strategies to induce V2 apex bnAb responses.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/imunologia , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/imunologia , Sítios de Ligação , Epitopos/imunologia , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/classificação , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/virologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Testes de Neutralização , Fragmentos de Peptídeos/imunologia , Filogenia , Ligação Proteica/imunologia , Conformação Proteica , Multimerização ProteicaRESUMO
The Ser/Thr protein kinase mTOR controls metabolic pathways, including the catabolic process of autophagy. Autophagy plays additional, catabolism-independent roles in homeostasis of cytoplasmic endomembranes and whole organelles. How signals from endomembrane damage are transmitted to mTOR to orchestrate autophagic responses is not known. Here we show that mTOR is inhibited by lysosomal damage. Lysosomal damage, recognized by galectins, leads to association of galectin-8 (Gal8) with the mTOR apparatus on the lysosome. Gal8 inhibits mTOR activity through its Ragulator-Rag signaling machinery, whereas galectin-9 activates AMPK in response to lysosomal injury. Both systems converge upon downstream effectors including autophagy and defense against Mycobacterium tuberculosis. Thus, a novel galectin-based signal-transduction system, termed here GALTOR, intersects with the known regulators of mTOR on the lysosome and controls them in response to lysosomal damage. VIDEO ABSTRACT.
Assuntos
Autofagia , Galectinas/metabolismo , Lisossomos/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Tuberculose/enzimologia , Proteínas Quinases Ativadas por AMP/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Galectinas/deficiência , Galectinas/genética , Células HEK293 , Células HeLa , Humanos , Lisossomos/microbiologia , Lisossomos/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos , Mycobacterium tuberculosis/patogenicidade , Transdução de Sinais , Células THP-1 , Serina-Treonina Quinases TOR/genética , Tuberculose/genética , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
In this study, we advance our understanding of the spatial relationship between the purinosome, a liquid condensate consisting of six enzymes involved in de novo purine biosynthesis, and mitochondria. Previous research has shown that purinosomes move along tubulin toward mitochondria, suggesting a direct uptake of glycine from mitochondria. Here, we propose that the purinosome is located proximally to the mitochondrial transporters SLC25A13 and SLC25A38, facilitating the uptake of glycine, aspartate, and glutamate, essential factors for purine synthesis. We utilized the proximity ligation assay (PLA) and APEX proximity labeling to investigate the association between purinosome proteins and mitochondrial transporters. Our results indicate that purinosome assembly occurs close to the mitochondrial membrane under purine-deficient conditions, with the transporters migrating to be adjacent to the purinosome. Furthermore, both targeted and non-targeted analyses suggest that the SLC25A13-APEX2-V5 probe accurately reflects endogenous cellular status. These findings provide insights into the spatial organization of purine biosynthesis and lay the groundwork for further investigations into additional proteins involved in this pathway.
RESUMO
Successful B cell activation, which is critical for high-affinity antibody production, is controlled by the B cell antigen receptor (BCR). However, we still lack a comprehensive protein-level view of the very dynamic multi-branched cellular events triggered by antigen binding. Here, we employed APEX2 proximity biotinylation to study antigen-induced changes, 5-15â min after receptor activation, at the vicinity of the plasma membrane lipid rafts, wherein BCR enriches upon activation. The data reveals dynamics of signaling proteins, as well as various players linked to the subsequent processes, such as actin cytoskeleton remodeling and endocytosis. Interestingly, our differential expression analysis identified dynamic responses in various proteins previously not linked to early B cell activation. We demonstrate active SUMOylation at the sites of BCR activation in various conditions and report its functional role in BCR signaling through the AKT and ERK1/2 axes.
Assuntos
Linfócitos B , Proteômica , Sumoilação , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
Differentiation of stem cells in the plant apex gives rise to aerial tissues and organs. Presently, we lack a lineage map of the shoot apex cells in woody perennials - a crucial gap considering their role in determining primary and secondary growth. Here, we used single-nuclei RNA-sequencing to determine cell type-specific transcriptomes of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of the epidermis, leaf mesophyll and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we applied a pipeline for interspecific single-cell gene expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the principles underlying cell division and differentiation conserved between herbaceous and perennial species while also allowing us to examine species-specific differences at single-cell resolution.
Assuntos
Arabidopsis , Populus , Arabidopsis/genética , Arabidopsis/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Populus/genética , Populus/metabolismo , RNA/metabolismo , Transcriptoma/genética , Xilema/metabolismoRESUMO
Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.