RESUMO
Targeted protein degradation using degrons, such as the mini-Auxin-inducible degron (mAID), has an advantage over genetic silencing/knockout. However, the efficiency of sgRNA, homologous recombination, tedious expansion, and screening single clones makes the process of tagging endogenous proteins long and laborious. This protocol describes a practical and economical way to obtain AID-tagged endogenous proteins using CRISPR/Cas9-mediated homology-directed repair (HDR). We use the generation of endogenously AID-tagged SPT6 in U2OS cells as an example but provide sufficient details for usage in other cell types. For complete details on the use and execution of this protocol, please refer to Narain et al. (2021).
Assuntos
Clonagem Molecular/métodos , Técnicas de Introdução de Genes/métodos , Proteínas de Plantas/genética , Proteólise , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Humanos , RNA Guia de Cinetoplastídeos/genética , TransfecçãoRESUMO
SPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor.
Assuntos
RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo , Linhagem Celular , Replicação do DNA , Humanos , Ácidos Indolacéticos/farmacologia , Poliadenilação , Proteólise/efeitos dos fármacos , RNA/biossíntese , RNA Polimerase II/genética , Fatores de Transcrição/genéticaRESUMO
The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.
Assuntos
Antineoplásicos/química , Aurora Quinase A/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteólise/efeitos dos fármacos , Talidomida/química , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Aurora Quinase A/genética , Benzazepinas/química , Domínio Catalítico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Desenho de Fármacos , Feminino , Humanos , Masculino , Terapia de Alvo Molecular , Polietilenoglicóis/química , Ligação Proteica , Conformação ProteicaRESUMO
The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.
Assuntos
Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinases Ciclina-Dependentes/genética , Chaperonas de Histonas/genética , Humanos , Neoplasias/genética , Regiões Promotoras Genéticas , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Cell membrane coated nanoparticles (NPs) is a biomimetic strategy developed to engineer therapeutic devices consisting of a NP core coated with membrane derived from natural cells such as erythrocytes, white blood cells, cancer cells, stem cells, platelets or bacterial cells. These biomimetic NPs have gained a lot of attention recently owing to their cell surface mimetic features and tailored nanomaterial characteristics. They have shown strong potential in diagnostic and therapeutic applications including those in drug delivery, immune modulation, vaccination and detoxification. Herein we review the various types of cell membrane coated NPs reported in the literature and the unique strengths of these biomimetic NPs with an emphasis on how these bioinspired camouflage strategies have led to improved therapeutic efficacy. We also highlight the recent progress made by each platform in advancing healthcare and precis the major challenges associated with these NPs.
