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1.
Mol Cell Proteomics ; 20: 100128, 2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34332124

RESUMO

Understanding how proteins are organized in compartments is essential to elucidating their function. While proximity-dependent approaches such as BioID have enabled a massive increase in information about organelles, protein complexes, and other structures in cell culture, to date there have been only a few studies on living vertebrates. Here, we adapted proximity labeling for protein discovery in vivo in the vertebrate model organism, zebrafish. Using lamin A (LMNA) as bait and green fluorescent protein (GFP) as a negative control, we developed, optimized, and benchmarked in vivo TurboID and miniTurbo labeling in early zebrafish embryos. We developed both an mRNA injection protocol and a transgenic system in which transgene expression is controlled by a heat shock promoter. In both cases, biotin is provided directly in the egg water, and we demonstrate that 12 h of labeling are sufficient for biotinylation of prey proteins, which should permit time-resolved analysis of development. After statistical scoring, we found that the proximal partners of LMNA detected in each system were enriched for nuclear envelope and nuclear membrane proteins and included many orthologs of human proteins identified as proximity partners of lamin A in mammalian cell culture. The tools and protocols developed here will allow zebrafish researchers to complement genetic tools with powerful proteomics approaches.

2.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32763951

RESUMO

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Assuntos
Betacoronavirus/genética , Fases de Leitura Aberta/genética , Betacoronavirus/isolamento & purificação , COVID-19 , Clonagem Molecular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Humanos , Pandemias , Plasmídeos/genética , Plasmídeos/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Potyvirus/genética , SARS-CoV-2
3.
Nat Metab ; 2(6): 499-513, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32694731

RESUMO

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases, including cancer. Because cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to the loss of de novo fatty acid biosynthesis. Here, we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in fatty acid synthase (FASN), whose product catalyses the formation of long-chain fatty acids. FASN-mutant cells show a strong dependence on lipid uptake that is reflected in negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking and protein glycosylation. Further support for these functional relationships is derived from additional GI screens in query cell lines deficient in other genes involved in lipid metabolism, including LDLR, SREBF1, SREBF2 and ACACA. Our GI profiles also identify a potential role for the previously uncharacterized gene C12orf49 (which we call LUR1) in regulation of exogenous lipid uptake through modulation of SREBF2 signalling in response to lipid starvation. Overall, our data highlight the genetic determinants underlying the cellular adaptation associated with loss of de novo fatty acid synthesis and demonstrate the power of systematic GI mapping for uncovering metabolic buffering mechanisms in human cells.


Assuntos
Ácidos Graxos/biossíntese , Metabolismo dos Lipídeos/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Mapeamento Cromossômico , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Humanos , Lipogênese/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Inanição/genética , Inanição/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
4.
Biochim Biophys Acta Gen Subj ; 1863(11): 129415, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31404619

RESUMO

BACKGROUND: AID/APOBEC3 (A3) enzymes instigate genomic mutations that are involved in immunity and cancer. Although they can deaminate any deoxycytidine (dC) to deoxyuridine (dU), each family member has a signature preference determined by nucleotides surrounding the target dC. This WRC (W = A/T, R = A/G) and YC (Y = T/C) hotspot preference is established for AID and A3A/A3B, respectively. Base alkylation and oxidation are two of the most common types of DNA damage induced environmentally or by chemotherapy. Here we examined the activity of AID, A3A and A3B on dCs neighboring such damaged bases. METHODS: Substrates were designed to contain target dCs either in normal WRC/YC hotspots, or in oxidized/alkylated DNA motifs. AID, A3A and A3B were purified and deamination kinetics of each were compared between substrates containing damaged vs. normal motifs. RESULTS: All three enzymes efficiently deaminated dC when common damaged bases were present in the -2 or -1 positions. Strikingly, some damaged motifs supported comparable or higher catalytic efficiencies by AID, A3A and A3B than the WRC/YC motifs which are their most favored normal sequences. Based on the resolved interactions of AID, A3A and A3B with DNA, we modeled interactions with alkylated or oxidized bases. Corroborating the enzyme assay data, the surface regions that recognize normal bases are predicted to also interact robustly with oxidized and alkylated bases. CONCLUSIONS: AID, A3A and A3B can efficiently recognize and deaminate dC whose neighbouring nucleotides are damaged. GENERAL SIGNIFICANCE: Beyond AID/A3s initiating DNA damage, some forms of pre-existing damaged DNA can constitute favored targets of AID/A3s if encountered.


Assuntos
Citidina Desaminase/química , Dano ao DNA , Desoxicitidina/química , Antígenos de Histocompatibilidade Menor/química , Proteínas/química , Citidina Desaminase/metabolismo , Desaminação , Desoxicitidina/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor/metabolismo , Oxirredução , Proteínas/metabolismo
5.
Mol Cell ; 73(3): 621-638.e17, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30554943

RESUMO

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Antineoplásicos/química , Azepinas/química , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Células K562 , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/química
6.
Mol Cell Proteomics ; 17(11): 2256-2269, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29991506

RESUMO

Proximity-dependent biotinylation strategies have emerged as powerful tools to characterize the subcellular context of proteins in living cells. The popular BioID approach employs an abortive E. coli biotin ligase mutant (R118G; denoted as BirA*), which when fused to a bait protein enables the covalent biotinylation of endogenous proximal polypeptides. This approach has been mainly applied to the study of protein proximity in immortalized mammalian cell lines. To expand the application space of BioID, here we describe a set of lentiviral vectors that enable the inducible expression of BirA*-tagged bait fusion proteins for performing proximity-dependent biotinylation in diverse experimental systems. We benchmark this highly adaptable toolkit across immortalized and primary cell systems, demonstrating the ease, versatility and robustness of the system. We also provide guidelines to perform BioID using these reagents.


Assuntos
Técnicas de Transferência de Genes , Lentivirus/metabolismo , Animais , Biotinilação , Vetores Genéticos/metabolismo , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Transgenes
7.
Mol Immunol ; 93: 94-106, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29161581

RESUMO

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID.


Assuntos
Citidina Desaminase/metabolismo , DNA/metabolismo , Região de Troca de Imunoglobulinas , Conformação de Ácido Nucleico , RNA/metabolismo , Composição de Bases , Sequência de Bases , Catálise , Citidina Desaminase/química , Citidina Desaminase/isolamento & purificação , DNA de Cadeia Simples/metabolismo , Desaminação , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Hibridização de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Transcrição Genética
8.
Nucleic Acids Res ; 41(10): 5457-68, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23585279

RESUMO

Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure.


Assuntos
5-Metilcitosina/metabolismo , Citidina Desaminase/metabolismo , Desoxicitidina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Domínio Catalítico , Citidina Desaminase/química , DNA de Cadeia Simples/metabolismo , Humanos , Modelos Moleculares , Peixe-Zebra , Proteínas de Peixe-Zebra/química
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