RESUMEN
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.
Asunto(s)
Neuronas/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN , Factores de Transcripción/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones Noqueados , Neuronas/química , Fosforilación , Dominios Proteicos , ARN/química , ARN/genética , ARN/metabolismo , ARN Polimerasa II/genética , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig-like domain 20 of FLNc, is doubly phosphorylated. The protein kinases responsible for this dual-site phosphorylation are Akt and PKCα. Proximity proteomics and interaction analysis identified filamin A-interacting protein 1 (FILIP1) as direct FLNc binding partner. FILIP1 binding induces filamin degradation, thereby negatively regulating its function. Here, dual-site phosphorylation of FLNc not only reduces FILIP1 binding, providing a mechanism to shield FLNc from FILIP1-mediated degradation, but also enables fast dynamics of FLNc necessary for its function as signaling adaptor in cross-striated muscle cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Filaminas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Secuencias de Aminoácidos , Células HEK293 , Humanos , Desarrollo de Músculos , Fibras Musculares Esqueléticas/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Unión Proteica , Proteolisis , Proteoma/análisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
The crystallizable fragment (Fc) of the immunoglobulin class G (IgG) is an attractive scaffold for the design of novel therapeutics. Upon engineering the C-terminal loops in the CH3 domains, Fcabs (Fc domain with antigen-binding sites) can be designed. We present the first crystal structures of Fcabs, i.e., of the HER2-binding clone H10-03-6 having the AB and EF loop engineered and the stabilized version STAB19 derived by directed evolution. Comparison with the crystal structure of the Fc wild-type protein reveals conservation of the overall domain structures but significant differences in EF-loop conformations. Furthermore, we present the first Fcab-antigen complex structures demonstrating the interaction between the engineered Fcab loops with domain IV of HER2. The crystal structures of the STAB19-HER2 and H10-03-6-HER2 complexes together with analyses by isothermal titration calorimetry, SEC-MALS, and fluorescence correlation spectroscopy show that one homodimeric Fcab binds two HER2 molecules following a negative cooperative binding behavior.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/metabolismo , Sitios de Unión , Calorimetría/métodos , Cromatografía en Gel , Cristalografía por Rayos X , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Mutación , Conformación Proteica , Dominios Proteicos , Estabilidad Proteica , Espectrometría de Fluorescencia , Trastuzumab/química , Trastuzumab/metabolismoRESUMEN
Protein phosphorylation has important regulatory functions in cell homeostasis and is tightly regulated by kinases and phosphatases. The tegument of human cytomegalovirus (CMV) contains not only several proteins reported to be extensively phosphorylated but also cellular protein phosphatases (PP1 and PP2A). To investigate this apparent inconsistency, we evaluated the phosphorylation status of the tegument proteins pUL32 and pp65 by enzymatic dephosphorylation and MS. Enzymatic dephosphorylation with bacterial λ phosphatase, but not with PP1, shifted the pUL32-specific signal on reducing SDS-PAGE from ~150 to ~148 kDa, a mass still much larger than the ~118 kDa obtained from our diffusion studies and from the calculated protein mass of ~113 kDa. Remarkably, inhibition of phosphatases through treatment with the phosphatase inhibitors calyculin A and okadaic acid resulted in a shift to ~190 or ~180 kDa, respectively, indicating that a considerable number of potential phosphorylated residues on pUL32 are not phosphorylated under normal conditions. MS revealed a general state of hypophosphorylation of CMV phosphoproteins with only 17 phosphorylated residues detected on pUL32 and 19 on pp65, respectively. Moreover, bioinformatics analysis shows that the C-terminal two-thirds of pUL32 are intrinsically disordered and that most phosphorylations map to this region. In conclusion, we show that important CMV tegument proteins are indeed phosphorylated, though to a lesser extent than previously reported, and the difference in mobility on SDS-PAGE and calculated mass of pUL32 may not be attributed to phosphorylation but more likely due to the partially intrinsically disordered nature of pUL32.
Asunto(s)
Citomegalovirus/metabolismo , Fosfoproteínas/metabolismo , Proteínas de la Matriz Viral/metabolismo , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Humanos , Toxinas Marinas , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
BACKGROUND: The hypothalamic-pituitary-adrenal axis (HPA axis) is a major part of the neuroendocrine system responsible for the regulation of the response to physical or mental stress and for the control of the synthesis of the stress hormone cortisol. Dysfunctions of the HPA axis characterized by either low (hypocortisolism) or increased (hypercortisolism) cortisol levels are implicated in various pathological conditions. Their understanding and therapeutic correction may be supported by mathematical modeling and simulation of the HPA axis. METHODS: Mass action and Michaelis Menten enzyme kinetics were used to provide a mechanistic description of the feedback mechanisms within the pituitary gland cells by which cortisol inhibits its own production. A separation of the nucleus from the cytoplasm by compartments enabled a differentiation between slow genomic and fast non-genomic processes. The model in parts was trained against time resolved ACTH stress response data from an in vitro cell culture of murine AtT-20 pituitary tumor cells and analyzed by bifurcation discovery tools. RESULTS: A recently found pituitary gland cell membrane receptor that mediates rapid non-genomic actions of glucocorticoids has been incorporated into our model of the HPA axis. As a consequence of the distinction between genomic and non-genomic feedback processes our model possesses an extended dynamic repertoire in comparison to existing HPA models. In particular, our model exhibits limit cycle oscillations and bistable behavior associated to hypocortisolism but also features a (second) bistable switch which captures irreversible transitions in hypercortisolism to elevated cortisol levels. CONCLUSIONS: Model predictive control and inverse bifurcation analysis have been previously applied in the simulation-based design of therapeutic strategies for the correction of hypocortisolism. Given the HPA model extension presented in this paper, these techniques may also be used in the study of hypercortisolism. As an example, we show how sparsity enforcing penalization may suggest network interventions that allow the return from elevated cortisol levels back to nominal ones.