RESUMO
ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADPRylation. Recently we developed a set of recombinant antibody-like ADP-ribose (ADPR) binding proteins using naturally occurring ADPR binding domains (ARBDs), including macrodomains and WWE domains, functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with the sequence from two other species, mouse and goat. These new reagents are based on a previously characterized set of naturally occurring ARBDs with known specificity. Characterization of the new reagents demonstrates that they can be detected in a species-dependent manner by secondary immunological tools, recognize specific ADPR moieties, and can be used for simultaneous detection of mono ADPR and poly ADPR at single-cell resolution in various antibody-based assays. The expansion of this toolkit will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems.
RESUMO
ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADP-ribosylation. Recently we developed a set of recombinant antibody-like ADP-ribose binding proteins, using naturally occurring ADPR binding domains (ARBDs) that include macrodomains and WWE domains, that have been functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with two other species, the Fc for mouse and goat immunogloblulin. Characterization of the new reagents indicates that they can be detected in a species-dependent manner, recognize specific ADP-ribose moieties, and excitingly, can be used in various antibody-based assays by co-staining. The expansion of this tool will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems.
RESUMO
Estradiol (E2) and relaxin (Rln) are steroid and polypeptide hormones, respectively, with important roles in the female reproductive tract, including myometrium. Some actions of Rln, which are mediated by its membrane receptor RXFP1, require or are augmented by E2 signaling through its cognate nuclear steroid receptor, estrogen receptor alpha (ERα). In contrast, other actions of Rln act in opposition to the effects of E2. Here we explore the molecular and genomic mechanisms that underlie the functional interplay between E2 and Rln in the myometrium. We used both ovariectomized female mice and immortalized human myometrial cells expressing wild type or mutant ERα (hTERT-HM-ERα cells). Our results indicate that Rln attenuates the genomic actions and biological effects of estrogen in the myometrium and myometrial cells by reducing phosphorylation ERα on serine 118 (S118). Interestingly, we observed a potent inhibitory effect of Rln on the E2-dependent binding of ERα across the genome. The reduction in ERα binding was associated with changes in the hormone-regulated transcriptome, including a decrease in the E2-dependent expression of neighboring genes. The inhibitory effects of Rln cotreatment on the E2-dependent phosphorylation of ERα required the nuclear dual-specificity phosphatases DUSP1 and DUSP5. Moreover, the inhibitory effects of Rln were reflected in a concomitant inhibition of the E2-dependent contraction of myometrial cells. Collectively, our results identify a pathway that integrates Rln/RXFP1 and E2/ERα signaling, resulting in a convergence of membrane and nuclear signaling pathways to control genomic and biological outcomes.
RESUMO
The demand for health care has increased with the development of global technology and the rise of public health awareness, and smart service systems have also been introduced to medical care to relieve the pressure on hospital staff. However, the survey found that patients' willingness to use smart services at the time of consultation has not improved. The main research purpose of this study was to understand the willingness of patients from various groups to use smart medical service systems and to explore the influencing factors on patients' use of smart service systems in hospitals through the technology acceptance model. This study distributed questionnaires in the outpatient area of National Taiwan University Hospital Yunlin Branch, and a total of 202 valid questionnaires were obtained. After related research and regression analysis, it was found that patients paid more attention to the benefits and convenience brought by smart services. If patients believed that smart services were trustworthy and beneficial to themselves, their usage intention and attitude would be positive. The results of this study are summarized by the following four points: (1) Designed according to the cultural conditions of different regions; (2) think about design from the patient's perspective; (3) strengthen the explanation and promotion of smart services; and (4) add humanized care and design. This study could be used as a reference for hospitals to improve their service quality and systems in the future.
Assuntos
Hospitais , Intenção , Humanos , Saúde Pública , Inquéritos e Questionários , TecnologiaRESUMO
Recent studies have identified cancer-associated mutations in histone genes that lead to the expression of mutant versions of core histones called oncohistones. Many oncohistone mutations occur at Asp and Glu residues, two amino acids known to be ADP-ribosylated (ADPRylated) by PARP1. We screened 25 Glu or Asp oncohistone mutants for their effects on cell growth in breast and ovarian cancer cells. Ectopic expression of six mutants of three different core histones (H2B, H3, and H4) altered cell growth in at least two different cell lines. Two of these sites, H2B-D51 and H4-D68, were indeed sites of ADPRylation in wild-type (unmutated) histones, and mutation of these sites inhibited ADPRylation. Mutation of H2B-D51 dramatically altered chromatin accessibility at enhancers and promoters, as well as gene expression outcomes, whereas mutation of H4-D68 did not. Additional biochemical, cellular, proteomic, and genomic analyses demonstrated that ADPRylation of H2B-D51 inhibits p300-mediated acetylation of H2B at many Lys residues. In breast cancer cell xenografts in mice, H2B-D51A promoted tumor growth, but did not confer resistance to the cytotoxic effects of PARP inhibition. Collectively, these results demonstrate that functional Asp and Glu ADPRylation sites on histones are mutated in cancers, allowing cancer cells to escape the growth-regulating effects of post-translational modifications via distinct mechanisms. SIGNIFICANCE: This study identifies cancer-driving mutations in histones as sites of PARP1-mediated ADP-ribosylation in breast and ovarian cancers, providing a molecular pathway by which cancers may subvert the growth-regulating effects of PARP1.