FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme's biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown.
View Article and Find Full Text PDFThe function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown.
View Article and Find Full Text PDFStatins are a mainstay intervention for cardiovascular disease prevention, yet their use can cause rare severe myopathy. HMG-CoA reductase, an essential enzyme in the mevalonate pathway, is the target of statins. We identified nine individuals from five unrelated families with unexplained limb-girdle like muscular dystrophy and bi-allelic variants in HMGCR via clinical and research exome sequencing.
View Article and Find Full Text PDFMammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the protein's native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent.
View Article and Find Full Text PDFRNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription.
View Article and Find Full Text PDFThe majority of in vitro studies of living cells are routinely conducted in a two-dimensional (2D) monolayer culture. Recent studies, however, suggest that 2D cell culture promotes specific types of aberrant cell behaviors due to the growth on non-physiologically stiff surfaces and the lack of the tissue-based extracellular matrix. Here, we investigate the sensitivity of the two-photon (2P) rotational dynamics of the intrinsic reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, to changes in the metabolic state of the metastatic murine breast cancer cells (4T1) in 2D monolayer and three-dimensional (3D) collagen matrix cultures.
View Article and Find Full Text PDFMost studies using intrinsic NAD(P)H as biomarkers for energy metabolism and mitochondrial anomalies have been conducted in routine two-dimensional (2D) cell culture formats. Cellular metabolism and cell behavior, however, can be significantly different in 2D cultures from that in vivo. As a result, there are emerging interests in integrating noninvasive, quantitative imaging techniques of NAD(P)H with in vivo-like three-dimensional (3D) models.
View Article and Find Full Text PDFLiving cells are crowded with dynamic distributions of macromolecules and organelles that influence protein diffusion, molecular transport, biochemical reactions, and protein assembly. Here, we test the hypothesis that the diffusion of single molecules deviates from Brownian motion as described by the Stokes-Einstein model in a manner that depends on the viscosity range, the chemical structure of both the diffusing species and the crowding agents, and the spatio-temporal resolution of the employed analytical methods. Our size-dependent fluorescent probes are rhodamine-110, quantum dots, enhanced green fluorescent proteins (EGFP), and mCerulean3-linker-mCitrine FRET probes with various linker length and flexibility.
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