RESUMEN
Signal transduction pathways are responsible for maintaining cellular functions, including proliferation, differentiation, apoptosis, and cell cycle progression. These pathways are maintained through the propagation of phosphorylation signals by protein kinases, as well as the removal of phosphorylation signals by protein phosphatases. Depending on the context, post-translational modification could have either a positive or negative effect on a signaling pathway. Intricate networks of positive and negative regulators offer a challenging target for the dissection of cell signaling mechanisms, particularly regarding the more subtle dampening of signal transduction through phosphatases. We report the development of two complimentary methods for the optical control of a complex phosphatase: SH2 domain-containing protein tyrosine phosphatase-2 (SHP2). We investigated controlling the catalytic function of SHP2 through (1) site-specific incorporation of a caged tyrosine for light activation of catalytic activity for the control of an essential substrate binding residue and (2) site-specific incorporation of a caged lysine at a conserved residue within an allosteric pocket for the control of SHP2 binding partner docking sites. These methods are generalizable to proteins bearing either a protein tyrosine phosphatase (PTP) catalytic domain or an SH2 domain, including SHP1, PTP family phosphatases, and a diverse range of SH2 domain-containing proteins.
Asunto(s)
Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Dominios Homologos src , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteínas Tirosina Fosfatasas con Dominio SH2 , Transducción de Señal , FosforilaciónRESUMEN
Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene (ADH) pathway serve to leverage consecutive kinases to convert exogenous alcohols into pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway-isopentenyl phosphate kinase from Thermoplasma acidophilum-towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide unnatural pyrophosphates and provide a critical first step in characterizing the rate-limiting steps in the artificial ADH pathway.
Asunto(s)
Hemiterpenos/síntesis química , Proteínas Quinasas/metabolismo , Especificidad por Sustrato , Terpenos/síntesis química , Thermoplasma/enzimología , Alcoholes , Difosfatos/metabolismo , Fosfatos/metabolismo , Biología Sintética/métodosRESUMEN
Rapamycin-induced dimerization of FKBP and FRB has been utilized as a tool for co-localizing two proteins of interest in numerous applications. Due to the tight binding interaction of rapamycin with FKBP and FRB, the ternary complex formation is essentially irreversible. Since biological processes occur in a highly dynamic fashion with cycles of protein association and dissociation to generate a cellular response, it is useful to have chemical tools that function in a similar manner. We have developed arylazopyrazole-modified rapamycin analogs which undergo a configurational change upon light exposure and we observed enhanced ternary complex formation for the cis-isomer over the trans-isomer for one of the analogs.
Asunto(s)
Compuestos Azo/química , Luz , Pirazoles/química , Sirolimus/química , Compuestos Azo/síntesis química , Células HEK293 , Humanos , Estructura Molecular , Pirazoles/síntesis química , Sirolimus/síntesis química , EstereoisomerismoRESUMEN
We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.
Asunto(s)
Dimerización , Endopeptidasas/química , Tetrahidrofolato Deshidrogenasa/química , Compartimento Celular/efectos de los fármacos , Compartimento Celular/genética , Compartimento Celular/efectos de la radiación , Luz , Orgánulos/química , Orgánulos/efectos de la radiación , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Transporte de Proteínas/efectos de la radiación , Sirolimus/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tetrahidrofolato Deshidrogenasa/efectos de la radiaciónRESUMEN
Biological processes are naturally regulated with high spatial and temporal control, as is perhaps most evident in metazoan embryogenesis. Chemical tools have been extensively utilized in cell and developmental biology to investigate cellular processes, and conditional control methods have expanded applications of these technologies toward resolving complex biological questions. Light represents an excellent external trigger since it can be controlled with very high spatial and temporal precision. To this end, several optically regulated tools have been developed and applied to living systems. In this review we discuss recent developments of optochemical tools, including small molecules, peptides, proteins, and nucleic acids that can be irreversibly or reversibly controlled through light irradiation, with a focus on applications in cells and animals.
Asunto(s)
Fenómenos Fisiológicos Celulares/efectos de la radiación , Optogenética/métodos , Fotoquímica/métodos , Animales , Fenómenos Fisiológicos Celulares/efectos de los fármacos , Descubrimiento de Drogas/métodos , Humanos , Luz , Simulación del Acoplamiento Molecular , Ácidos Nucleicos/genética , Ácidos Nucleicos/metabolismo , Péptidos/genética , Péptidos/metabolismo , Procesos Fotoquímicos , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/efectos de la radiación , Proteínas/genética , Proteínas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
An arylazopyrazole was explored for its use as an enhanced photoswitchable amino acid in genetic code expansion. This new unnatural amino acid was successfully incorporated into proteins in both bacterial and mammalian cells. While photocontrol of translation required pulsed irradiations, complete selectivity for the trans-configuration by the pyrrolysyl tRNA synthetase was observed, demonstrating expression of a gene of interest selectively controlled via light exposure.
RESUMEN
Chemically induced dimerization of FKBP and FRB using rapamycin and rapamycin analogs has been utilized in a variety of biological applications. Formation of the FKBP-rapamycin-FRB ternary complex is typically used to activate a biological process and this interaction has proven to be essentially irreversible. In many cases, it would be beneficial to also have temporal control over deactivating a biological process once it has been initiated. Thus, we developed the first reactive oxygen species-generating rapamycin analog toward this goal. The BODIPY-rapamycin analog BORap is capable of dimerizing FKBP and FRB to form a ternary complex, and upon irradiation with 530 nm light, generates singlet oxygen to oxidize and inactivate proteins of interest fused to FKBP/FRB.
RESUMEN
Rapamycin-induced dimerization of FKBP and FRB is the most commonly utilized chemically induced protein dimerization system. It has been extensively used to conditionally control protein localization, split-enzyme activity, and protein-protein interactions in general by simply fusing FKBP and FRB to proteins of interest. We have developed a new aminonitrobiphenylethyl caging group and applied it to the generation of a caged rapamycin analog that can be photoactivated using blue light. Importantly, the caged rapamycin analog shows minimal background activity with regard to protein dimerization and can be directly interfaced with a wide range of established (and often commercially available) FKBP/FRB systems. We have successfully demonstrated its applicability to the optical control of enzymatic function, protein stability, and protein subcellular localization. Further, we also showcased its applicability toward optical regulation of cell signaling, specifically mTOR signaling, in cells and aquatic embryos.
Asunto(s)
Luz , Proteínas/metabolismo , Sirolimus/análogos & derivados , Pez Cebra/embriología , Animales , Dimerización , Embrión no Mamífero/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Sirolimus/efectos de la radiación , Fracciones Subcelulares/metabolismoRESUMEN
Protein phosphatases play an essential role in cell signaling; however, they remain understudied compared with protein kinases, in part due to a lack of appropriate tools. In order to provide conditional control over phosphatase function, we developed two different approaches for rendering MKP3 (a dual-specific phosphatase, also termed DUSP6) activatable by light. Specifically, we expressed the protein with strategically placed light-removable protecting groups in cells with an expanded genetic code. This allowed for the acute perturbation of the Ras/MAPK signaling pathway upon photoactivation in live cells. In doing so, we confirmed that MKP3 does not act as a thresholding gate for growth factor stimulation of the extracellular signal-regulated kinase (ESRK) pathway.
Asunto(s)
Fosfatos , Fosfoproteínas Fosfatasas , Proteínas Quinasas , Transducción de SeñalRESUMEN
In this issue of Cell Chemical Biology, the Sterner lab reports the application of photoswitchable unnatural amino acids in controlling protein allostery in the enzyme complex imidazole glycerol phosphate synthase (Kneuttinger et al., 2019). Remarkable switching in enzyme activity was achieved upon photoisomerization of an azobenzene amino acid.
Asunto(s)
AminoácidosRESUMEN
Protein phosphatases are involved in embryonic development, metabolic homeostasis, stress response, cell cycle transitions, and many other essential biological mechanisms. Unlike kinases, protein phosphatases remain understudied and less characterized. Traditional genetic and biochemical methods have contributed significantly to our understanding; however, these methodologies lack precise and acute spatiotemporal control. Here, we report the development of a light-activated protein phosphatase, the dual specificity phosphatase 6 (DUSP6 or MKP3). Through genetic code expansion, MKP3 is placed under optical control via two different approaches: (i) incorporation of a caged cysteine into the active site for controlling catalytic activity and (ii) incorporation of a caged lysine into the kinase interaction motif for controlling the protein-protein interaction between the phosphatase and its substrate. Both strategies are expected to be applicable to the engineering of a wide range of light-activated phosphatases. Applying the optogenetically controlled MKP3 in conjunction with live cell reporters, we discover that ERK nuclear translocation is regulated in a graded manner in response to increasing MKP3 activity.
Asunto(s)
Fosfatasa 6 de Especificidad Dual/metabolismo , Activación Enzimática/efectos de la radiación , Proteínas Luminiscentes/metabolismo , Optogenética/métodos , Rayos Ultravioleta , Fosfatasa 6 de Especificidad Dual/química , Fosfatasa 6 de Especificidad Dual/genética , Células HEK293 , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Mutación , Imagen de Lapso de Tiempo/métodosRESUMEN
In nature, biological processes are regulated with precise spatial and temporal resolution at the molecular, cellular, and organismal levels. In order to perturb and manipulate these processes, optically controlled chemical tools have been developed and applied in living systems. The use of light as an external trigger provides spatial and temporal control with minimal adverse effects. Incorporation of light-responsive amino acids into proteins in cells and organisms with an expanded genetic code has enabled the precise activation/deactivation of numerous, diverse proteins, such as kinases, nucleases, proteases, and polymerases. Using unnatural amino acids to generate light-triggered proteins enables a rational engineering approach that is based on mechanistic and/or structural information. This review focuses on the most recent developments in the field, including technological advances and biological applications.