RESUMO
Statins 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. The clinical features resembled other genetic causes of muscular dystrophy with incidental high CPK levels (>1,000 U/L), proximal muscle weakness, variable age of onset, and progression leading to impaired ambulation. Muscle biopsies in most affected individuals showed non-specific dystrophic changes with non-diagnostic immunohistochemistry. Molecular modeling analyses revealed variants to be destabilizing and affecting protein oligomerization. Protein activity studies using three variants (p.Asp623Asn, p.Tyr792Cys, and p.Arg443Gln) identified in affected individuals confirmed decreased enzymatic activity and reduced protein stability. In summary, we showed that individuals with bi-allelic amorphic (i.e., null and/or hypomorphic) variants in HMGCR display phenotypes that resemble non-genetic causes of myopathy involving this reductase. This study expands our knowledge regarding the mechanisms leading to muscular dystrophy through dysregulation of the mevalonate pathway, autoimmune myopathy, and statin-induced myopathy.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Doenças Musculares , Distrofia Muscular do Cíngulo dos Membros , Distrofias Musculares , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Ácido Mevalônico , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Doenças Musculares/genética , Oxirredutases , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/efeitos adversosRESUMO
Phenomenon: To increase racial diversity in medical school classes, many institutions have created underrepresented minority (URM) application streams. However, many URM students experience overt and passive marginalization throughout their training and this may be related to how matriculants from URM streams are perceived by their peers. Approach: We conducted a discourse analysis of online discussion forums to explore how URM streams across Canada and the United States are perceived. We analyzed 850 posts from 13 discussion threads published between 2015 and 2020. We used inductive content analysis to develop a data-driven coding scheme from which we identified common themes. Findings: Despite an overall appreciation of the benefits of a diverse workforce, participants engaged in prominent discussions surrounding the merits of URM streams. We identified perceptions that students admitted from URM streams are less academically and clinically competent, with URM applicants reporting feeling unworthy for admission in the eyes of non-URM applicants. Users felt that the influence of socioeconomic status was under-appreciated, and that admissions officers inadequately addressed this barrier. There were some applicants who perceived the admissions process as "broken" with non-URMs displaying a fear of social change, and URMs fearing that the system defines them by their racialized status. Insights: Online discussion forums provide unique insight into perceptions surrounding URM streams. We identified potentially harmful misconceptions about URM students applying to these streams and highlight that actionable measures to reduce marginalization against URM matriculants must begin before medical school.
RESUMO
The 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. Time-resolved 2P-associated anisotropy measurements reveal that the rotational dynamics of free and enzyme-bound NAD(P)H in 4T1 cells are correlated to changes in the metabolic state of 2D and 3D cell cultures. In addition to the type of cell culture, we also investigated the metabolic response of 4T1 cells to treatment with two metabolic inhibitors (MD1 and TPPBr). The statistical analyses of our results enabled us to identify which of the fitting parameters of the observed time-resolved associate anisotropy of cellular NAD(P)H were significantly sensitive to changes in the metabolic state of 4T1 cells. Using a black-box model, the population fractions of free and bound NAD(P)H were used to estimate the corresponding equilibrium constant and the standard Gibbs free energy changes that are associated with underlying metabolic pathways of 4T1 cells in 2D and 3D cultures. These rotational dynamics analyses are in agreement with the standard 2P-fluorescence lifetime imaging microscopy (FLIM) measurements on the same cell line, cell cultures, and metabolic inhibition. These studies represent an important step towards the development of a noninvasive, time-resolved associated anisotropy to complement 2P-FLIM in order to elucidate the underlying cellular metabolism and metabolic plasticity in more complex in vivo, tumor-like models using intrinsic NADH autofluorescence.
Assuntos
Neoplasias da Mama/metabolismo , Colágeno/metabolismo , NADP/metabolismo , Animais , Anisotropia , Neoplasias da Mama/patologia , Colágeno/química , Feminino , Camundongos , NADP/análise , Células Tumorais CultivadasRESUMO
Most 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. The overall features and metabolic responses of the murine breast cancer cells line 4T1 in 2D cultures were compared with those in 3D collagen matrix using integrated optical micro-spectroscopy. The metabolic responses to two novel compounds, MD1 and TPPBr, that target metabolism by disrupting monocarboxylate transporters or oxidative phosphorylation (OXPHOS), respectively, were investigated using two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intracellular NAD(P)H in 2D and 3D cultures. 4T1 cells exhibit distinct behaviors in a collagenous 3D matrix from those in 2D culture, forming anastomosing multicellular networks and spherical acini in 3D culture, as opposed to simple flattened epithelial plaques in 2D culture. The cellular NAD(P)H in 3D collagen matrix exhibits a longer fluorescence lifetime as compared with 2D culture, which is attributed to an enhanced population of enzyme-bound NAD(P)H in the 3D culture. TPPBr induces mitochondrial hyperpolarization in 2D culture of 4T1 cells along with an enhanced free NAD(P)H population, which suggest an interference with OXPHOS. In contrast, 2P-FLIM of cellular NAD(P)H revealed an enhanced autofluorescence lifetime in 3D 4T1 cultures after MD1 treatment as compared with MD1-treated 2D culture and the control 3D culture. Physical and chemical microenvironmental signaling are critical factors in understanding how therapeutic compounds target cancer cells by disrupting their metabolic pathways. Integrating 2P-FLIM of intrinsic NAD(P)H with refined 3D tumor-matrix in vitro models promises to advance our understanding of the roles of metabolism and metabolic plasticity in tumor growth and metastatic behavior. © 2018 International Society for Advancement of Cytometry.
Assuntos
Neoplasias Mamárias Experimentais/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , NAD/metabolismo , Animais , Linhagem Celular Tumoral , Colágeno , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Feminino , Fluorescência , Neoplasias Mamárias Experimentais/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Fosforilação Oxidativa/efeitos dos fármacosRESUMO
Living 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. Using fluorescence correlation spectroscopy (FCS), we investigated the translational diffusion of structure-dependent fluorescent probes, at the single-molecule level, in homogeneous (glycerol) and heterogeneous (Ficoll-70) solutions as a function of the bulk viscosity. Complementary rotational diffusion studies using time-resolved anisotropy enable us to assess weak interactions in crowded and viscous environments. Overall, our results show negative deviation from the Stokes-Einstein model in a fluorophore- and environment-dependent manner. In addition, the deviation between the FCS-measured hydrodynamic radius of the FRET probes in a buffer at room temperature and the molecular-weight based estimate (Perrin equation) as the number of the amino acid residues in the linker increases. These studies are essential for quantitative biophysics using fluorescence- and diffusion-based studies of protein-protein interactions and biomolecular transport in living cells.
Assuntos
Corantes Fluorescentes/química , Difusão , Hidrodinâmica , Tamanho da Partícula , Rotação , Espectrometria de FluorescênciaRESUMO
Industrial accidents, wars and terrorist threats are potential sources of skin contamination by highly toxic chemical warfare agents and manufacturing compounds. We have compared the time-dependent adsorption capacity and decontamination efficiency of fuller's earth (FE) for four different formulations for the molecular tracer, 4-cyanophenol (4-CP), in vitro and ex vivo using water decontamination as standard. The adsorption capacity of FE was assessed in vitro for 4-CP aqueous solutions whereas decontamination efficiency was investigated ex vivo by tracking porcine skin 4-CP content using attenuated total reflectance Fourier transform infrared spectroscopy. Decontamination was performed on short time, exposed porcine skin to 4-CP by application of FE: (1) as free powder; (2) loaded on adhesive tape; (3) on powdered glove; or (4) in suspension. Removal rate of 4-CP from aqueous solutions correlates with the amount of FE and its contact time. Decontamination efficiency estimated by the percentage of 4-CP recovery from contaminated porcine skin, achieved 54% with water, ranged between ~60 and 70% with dry FE and reached ~90% with FE suspension. Successful decontamination of the FE suspension, enabling a dramatic reduction of skin contamination after a brief exposure scenario, appears to be rapid, reliable and should be formulated in a new device ready to use for self-application.
Assuntos
Compostos de Alumínio/farmacologia , Substâncias para a Guerra Química/toxicidade , Descontaminação/métodos , Compostos de Magnésio/farmacologia , Fenóis/toxicidade , Silicatos/farmacologia , Absorção Cutânea/efeitos dos fármacos , Pele/efeitos dos fármacos , Compostos de Alumínio/química , Animais , Substâncias para a Guerra Química/farmacocinética , Composição de Medicamentos , Técnicas In Vitro , Compostos de Magnésio/química , Fenóis/farmacocinética , Silicatos/química , Pele/metabolismo , SuínosRESUMO
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. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A's function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.
Assuntos
Serina Endopeptidases , Serina Proteases , Dimerização , Serina Endopeptidases/metabolismo , Proteólise , Replicação do DNA , SerinaRESUMO
The 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. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.
Assuntos
Genes Precoces , Proteína Quinase 1 Ativada por Mitógeno , Humanos , DNA/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Etoposídeo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Ativação TranscricionalRESUMO
Mammalian 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. Here, we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X-ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed.
Assuntos
Anticorpos de Domínio Único , Animais , Cromatografia de Afinidade , Cristalografia por Raios X , Humanos , Mamíferos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismoRESUMO
RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIß (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. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.
Assuntos
Proteína BRCA1/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/química , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Mutação , Fosforilação , Sítio de Iniciação de Transcrição , Transcrição Gênica , UbiquitinaçãoRESUMO
Rectangular V-amylose single crystals were prepared by adding racemic ibuprofen to hot dilute aqueous solutions of native and enzymatically-synthesized amylose. The lamellar thickness increased with increasing degree of polymerization of amylose and reached a plateau at about 7 nm, consistent with a chain-folding mechanism. The CP/MAS NMR spectrum as well as base-plane electron and powder X-ray diffraction patterns recorded from hydrated specimens were similar to those of V-amylose complexed with propan-2-ol. Amylose was crystallized in an orthorhombic unit cell with parameters a = 2.824 ± 0.001 nm, b = 2.966 ± 0.001 nm, and c = 0.800 ± 0.001 nm. A molecular model was proposed based on structural analogies with the Vpropan-2-ol complex and on assumptions on the stoichiometry of ibuprofen. The unit cell would contain four antiparallel 7-fold amylose single helices with ibuprofen molecules distributed inside and between the helices.
Assuntos
Amilose/química , Ibuprofeno/química , Nanopartículas/química , Varredura Diferencial de Calorimetria , Cristalização , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Estrutura Molecular , Nanoconjugados/química , Polimerização , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
The crystallization of amylose from dilute solutions in the presence of a series of linear saturated fatty acids (C3 to C20) was investigated by varying the fatty acid concentration, crystallization temperature and solvent composition (DMSO:water in various ratios). The morphology and structure of the resulting model lamellar crystals were characterized by transmission electron microscopy as well as electron and X-ray diffraction. By adequately controlling the crystallization parameters, all fatty acids could induce the formation of both 6- and 7-fold V-amylose single helices, indicating that the amylose conformation was independent of the chain length of the complexing molecule. Three allomorphs (V6I, V6II and V7) were identified individually or in mixtures. Higher concentrations of fatty acid and DMSO and a higher temperature promoted the formation of the more compact V6I structure. V6II and V7 preferentially formed with lower concentrations of fatty acids and DMSO and at lower temperatures. In the case of C5-C20 fatty acids, V7 was only obtained in the presence of DMSO. The polymorphism of V-amylose complexes with linear saturated fatty acids thus appears to be a more general phenomenon than previously reported in the literature.
Assuntos
Amilose/química , Ácidos Graxos/química , CristalizaçãoRESUMO
The use of robust ecological data to make evidence-based management decisions is frequently prevented by limited data quantity or quality, and local ecological knowledge (LEK) is increasingly seen as an important source of information for conservation. However, there has been little assessment of LEK's usefulness for informing prioritization and management of landscapes for threatened species, or assessing comparative species status across landscapes.A large-scale interview survey in the Annamite Mountains (Vietnam and Lao PDR) compiled the first systematic LEK data set for saola Pseudoryx nghetinhensis, one of the world's rarest mammals, and eight other ungulates. Saola conservation is hindered by uncertainty over continued presence across much of its proposed distribution. We analysed comparative LEK-based last-sighting data across three landscapes to determine whether regional sighting histories support previous suggestions of landscape importance for saola conservation (Hue-Quang Nam: top-priority Vietnamese landscape; Pu Mat: lower priority Vietnamese landscape; Viengthong: high-priority Lao landscape) and whether they constitute an effective spatial prioritization tool for cryptic species management.Wild pig and red muntjac may be the only Annamite ungulates with stable populations; the regional status of all other species appears to be worse. Saola have declined more severely and/or are significantly rarer than most other ungulates and have been seen by relatively few respondents. Saola were also frequently considered locally rarest or declining, and never as species that had not declined.In contrast to other species, there are no regional differences in saola sighting histories, with continued persistence in all landscapes challenging suggestions that regional status differs greatly. Remnant populations persist in Vietnam despite heavy hunting, but even remote landscapes in Lao may be under intense pressure.Synthesis and applications. Our local ecological knowledge data suggest that intact saola populations probably no longer exist, but individuals persist in all three landscapes, making management activities to reduce hunting pressure on ungulates in each landscape a conservation priority. Analysis of last-sighting histories can constitute an important conservation tool when robust data are otherwise unavailable, and collection of last-sighting records should be incorporated more widely into field studies and management of other highly threatened, cryptic species. Our local ecological knowledge data suggest that intact saola populations probably no longer exist, but individuals persist in all three landscapes, making management activities to reduce hunting pressure on ungulates in each landscape a conservation priority. Analysis of last-sighting histories can constitute an important conservation tool when robust data are otherwise unavailable, and collection of last-sighting records should be incorporated more widely into field studies and management of other highly threatened, cryptic species.