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1.
BMC Med Educ ; 24(1): 379, 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38589919

RESUMO

The United States government makes a substantial investment in biomedical training programs each year. However, for most trainees, these opportunities do not translate into career progression in academic research pathways. Only about one-fifth of postdoctoral fellows eventually secure a tenure-track faculty position, and even among these candidates, attrition is high. Although a number of factors govern career choices and career longevity, the transition from trainee to faculty is a challenging process and requires knowledge and skills that are not necessarily developed during a traditional university experience. Many postdoctoral fellows receive adequate training in research skills and scientific communication, but new faculty report not being sufficiently prepared for the job search process and for starting their labs. To address this critical training gap, the ITERT core (Interdisciplinary Translational Education and Research Training) and the Office of Postdoctoral Fellows at the University of Texas MD Anderson Cancer Center implemented a structured course for both postdoctoral fellows and senior PhD students to provide formalized training for successfully navigating academic positions in biomedical research. Here we report on the pilot Navigating Academic Careers course conducted in 2021-2022 for 30 PhD students and postdocs. The nine-module course was conducted over 13 weeks in 25.5 h instructional sessions. The key educational objectives included 1) navigating the job application and the interview/negotiation process, 2) hiring, leading, and mentoring lab personnel and program support staff, 3) project administration and financial stewardship, 4) managing time and work-life balance and 5) developing collaborations, branding, personalized niche, and networking. Survey-based analysis at the time of the course was used to capture the participants' assessment of the course content, organization, and delivery, with a follow-up survey conducted approximately 2 years post-course (2024) to evaluate longer-term impacts of the training. Initial in-course assessment revealed that 89.9% of respondents found the scope and instructional content appropriate, and 91.1% found the course relevant and applicable to their career needs. Longer-term post-course evaluation indicated that 80% of respondents applied the learnings of the course, that 80% reported feeling more confident in navigating an academic job search, and that 66.6% continued to report agreement with the course preparing them for their current role/ongoing job search, with 46.7% already securing jobs in academic research, including as independent faculty. The outcomes of this pilot course suggest that integrating this into the broader postdoctoral training curriculum can enhance both the transition and early-career success of talented scientists-in-training into working professionals in biomedical careers, as faculty and science-trained staff.


Assuntos
Pesquisa Biomédica , Tutoria , Humanos , Estados Unidos , Currículo , Docentes , Mentores , Escolha da Profissão
2.
Development ; 149(7)2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35297995

RESUMO

Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Remodelação Vascular , Saco Vitelino , Animais , Artérias , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Camundongos , Remodelação Vascular/genética , Saco Vitelino/metabolismo
3.
Methods Mol Biol ; 2319: 93-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331247

RESUMO

Lightsheet microscopy is a form of fluorescence microscopy that can be used to visualize specimen with high resolution, a large depth-of-field, and minimal photodamage and photobleaching as compared to traditional confocal microscopy. As this technology becomes much more readily available, it will be useful in revealing new findings in the cardiovascular development field that may be hidden or difficult to image. In this manuscript, we describe an approach for mounting and culturing postimplantation mouse embryos to visualize blood vessel development with a lightsheet microscope.


Assuntos
Angiografia/métodos , Vasos Sanguíneos/diagnóstico por imagem , Técnicas de Cultura/métodos , Embrião de Mamíferos/diagnóstico por imagem , Desenvolvimento Embrionário , Microscopia de Fluorescência/métodos , Neovascularização Fisiológica , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Meios de Cultura/química , Dissecação/métodos , Embrião de Mamíferos/irrigação sanguínea , Camundongos , Camundongos Transgênicos , Microscopia Confocal
4.
Mech Dev ; 156: 8-19, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30796970

RESUMO

Blood vessel maturation, which is characterized by the investment of vascular smooth muscle cells (vSMCs) around developing blood vessels, begins when vessels remodel into a hierarchy of proximal arteries and proximal veins that branch into smaller distal capillaries. The ultimate result of maturation is formation of the tunica media-the middlemost layer of a vessel that is composed of vSMCs and acts to control vessel integrity and vascular tone. Though many studies have implicated the role of various signaling molecules in regulating maturation, no studies have determined a role for hemodynamic force in the regulation of maturation in the mouse. In the current study, we provide evidence that a hemodynamic force-dependent mechanism occurs in the mouse because reduced blood flow mouse embryos exhibited a diminished or absent coverage of vSMCs around vessels, and in normal-flow embryos, extent of coverage correlated to the amount of blood flow that vessels were exposed to. We also determine that the cellular mechanism of force-induced maturation was not by promoting vSMC differentiation/proliferation, but instead involved the recruitment of vSMCs away from neighboring low-flow distal capillaries towards high-flow vessels. Finally, we hypothesize that hemodynamic force may regulate expression of specific signaling molecules to control vSMC recruitment to high-flow vessels, as reduction of flow results in the misexpression of Semaphorin 3A, 3F, 3G, and the Notch target gene Hey1, all of which are implicated in controlling vessel maturation. This study reveals another role for hemodynamic force in regulating blood vessel development of the mouse, and opens up a new model to begin elucidating mechanotransduction pathways regulating vascular maturation.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Músculo Liso Vascular/crescimento & desenvolvimento , Animais , Artérias/crescimento & desenvolvimento , Artérias/metabolismo , Vasos Sanguíneos/metabolismo , Proliferação de Células/genética , Hemodinâmica , Mecanotransdução Celular/genética , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo
5.
Dev Biol ; 442(1): 127-137, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30012423

RESUMO

Development of the embryonic heart involves an intricate network of biochemical and genetic cues to ensure its proper growth and morphogenesis. However, studies from avian and teleost models reveal that biomechanical force, namely hemodynamic loading (blood pressure and shear stress), plays a significant role in regulating heart development. To study how hemodynamic loading impacts development of the mammalian embryonic heart, we utilized mouse embryo culture and manipulation techniques and performed optical projection tomography imaging followed by morphometric analysis to determine how reduced-loading affects heart volume, myocardial thickness, trabeculation and looping. Our results reveal that hemodynamic loading can regulate these features at different thresholds. Intermediate levels of hemodynamic loading are sufficient to promote proper myocardial growth and heart size, but insufficient to promote looping and trabeculation. Whereas, low levels of hemodynamic loading fails to promote proper growth of the myocardium and heart size. These results reveal that the regulation of heart development by biomechanical force is conserved across many vertebrate classes, and this study begins to elucidate how these specific forces regulate development of the mammalian heart.


Assuntos
Coração/embriologia , Hemodinâmica/fisiologia , Animais , Fenômenos Biomecânicos/fisiologia , Camundongos/embriologia , Morfogênese/fisiologia , Miocárdio/patologia , Organogênese , Estresse Mecânico
6.
PLoS One ; 10(9): e0137175, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26340748

RESUMO

BACKGROUND: Vascular patterning depends on coordinated timing of arteriovenous specification of endothelial cells and the concomitant hemodynamic forces supplied by the onset of cardiac function. Using a combination of 3D imaging by OPT and embryo registration techniques, we sought to identify structural differences between three different mouse models of cardiovascular perturbation. RESULTS: Endoglin mutant mice shared a high degree of similarity to Mlc2a mutant mice, which have been shown to have a primary developmental heart defect causing secondary vessel remodeling failures. Dll4 mutant mice, which have well-characterized arterial blood vessel specification defects, showed distinct differences in vascular patterning when compared to the disruptions seen in Mlc2a-/- and Eng-/- models. While Mlc2a-/- and Eng-/- embryos exhibited significantly larger atria than wild-type, Dll4-/- embryos had significantly smaller hearts than wild-type, but this quantitative volume decrease was not limited to the developing atrium. Dll4-/- embryos also had atretic dorsal aortae and smaller trunks, suggesting that the cardiac abnormalities were secondary to primary arterial blood vessel specification defects. CONCLUSIONS: The similarities in Eng-/- and Mlc2a-/- embryos suggest that Eng-/- mice may suffer from a primary heart developmental defect and secondary defects in vessel patterning, while defects in Dll4-/- embryos are consistent with primary defects in vessel patterning.


Assuntos
Cardiopatias Congênitas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Miocárdio/metabolismo , Cadeias Leves de Miosina/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Artérias/crescimento & desenvolvimento , Artérias/metabolismo , Artérias/patologia , Proteínas de Ligação ao Cálcio , Embrião de Mamíferos , Endoglina , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Hemodinâmica , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Miocárdio/patologia , Cadeias Leves de Miosina/deficiência , Neovascularização Fisiológica/genética , Organogênese/genética
7.
Development ; 141(22): 4406-14, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25344073

RESUMO

Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as post-implantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants.


Assuntos
Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/fisiologia , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Imagem com Lapso de Tempo/métodos , Animais , Embrião de Mamíferos/citologia , Camundongos , Sefarose , Inclusão do Tecido/métodos
8.
Development ; 140(19): 4041-50, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24004946

RESUMO

Despite extensive work showing the importance of blood flow in angiogenesis and vessel remodeling, very little is known about how changes in vessel diameter are orchestrated at the cellular level in response to mechanical forces. To define the cellular changes necessary for remodeling, we performed live confocal imaging of cultured mouse embryos during vessel remodeling. Our data revealed that vessel diameter increase occurs via two distinct processes that are dependent on normal blood flow: vessel fusions and directed endothelial cell migrations. Vessel fusions resulted in a rapid change in vessel diameter and were restricted to regions that experience the highest flow near the vitelline artery and vein. Directed cell migrations induced by blood flow resulted in the recruitment of endothelial cells to larger vessels from smaller capillaries and were observed in larger artery segments as they expanded. The dynamic and specific endothelial cell behaviors captured in this study reveal how sensitive endothelial cells are to changes in blood flow and how such responses drive vascular remodeling.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Saco Vitelino/citologia , Saco Vitelino/embriologia , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Saco Vitelino/metabolismo
9.
Wiley Interdiscip Rev Dev Biol ; 2(3): 327-46, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23799579

RESUMO

The vasculature of an organism has the daunting task of connecting all the organ systems to nourish tissue and sustain life. This complex network of vessels and associated cells must maintain blood flow, but constantly adapt to acute and chronic changes within tissues. While the vasculature has been studied for over a century, we are just beginning to understand the processes that regulate its formation and how genetic hierarchies are influenced by mechanical and metabolic cues to refine vessel structure and optimize efficiency. As we gain insights into the developmental mechanisms, it is clear that the processes that regulate blood vessel development can also enable the adult to adapt to changes in tissues that can be elicited by exercise, aging, injury, or pathology. Thus, research in vessel development has provided tremendous insights into therapies for vascular diseases and disorders, cancer interventions, wound repair and tissue engineering, and in turn, these models have clearly impacted our understanding of development. Here we provide an overview of the development of the vascular system, highlighting several areas of active investigation and key questions that remain to be answered.


Assuntos
Sistema Cardiovascular/crescimento & desenvolvimento , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Humanos , Linfangiogênese , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
PLoS One ; 7(1): e30357, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22291941

RESUMO

The Transforming growth factor ß (Tgf-ß) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-ß signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta.


Assuntos
Amelogênese/genética , Esmalte Dentário/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Calcificação de Dente/genética , Animais , Calcificação Fisiológica/genética , Calcificação Fisiológica/fisiologia , Esmalte Dentário/crescimento & desenvolvimento , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Testes de Dureza , Integrases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad3/fisiologia , Doenças Dentárias/genética , Doenças Dentárias/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
11.
Dev Cell ; 22(2): 242-3, 2012 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-22340492

RESUMO

Recently, substantial advances have been made in understanding the formation and remodeling of the lymphatic vasculature. In this issue, Sabine et al. (2012) further define the mechanisms of lymphatic valve formation and implicate oscillatory shear stress in regulating the molecular events that control valve morphogenesis.

16.
Methods Enzymol ; 476: 329-49, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20691875

RESUMO

For the past three decades, methods for culturing mouse embryos ex vivo have been optimized in order to improve embryo viability and physiology throughout critical stages of embryogenesis. Combining advances made in the production of transgenic animals and in the development of different varieties of fluorescent proteins (FPs), time-lapse imaging is becoming more and more popular in the analysis of dynamic events during mouse development. Targeting FPs to specific cell types or subcellular compartments has enabled researchers to study cell proliferation, apoptosis, migration, and changes in cell morphology in living mouse embryos in real time. Here we provide a guide for time-lapse imaging of early stages of mouse embryo development.


Assuntos
Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Microscopia Confocal , Animais , Técnicas de Cultura Embrionária/instrumentação , Feminino , Corantes Fluorescentes/metabolismo , Masculino , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Gravidez , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem/métodos , Fatores de Tempo
17.
Genes Dev ; 21(21): 2747-61, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17974916

RESUMO

The Hippo pathway plays a key role in organ size control by regulating cell proliferation and apoptosis in Drosophila. Although recent genetic studies have shown that the Hippo pathway is regulated by the NF2 and Fat tumor suppressors, the physiological regulations of this pathway are unknown. Here we show that in mammalian cells, the transcription coactivator YAP (Yes-associated protein), is inhibited by cell density via the Hippo pathway. Phosphorylation by the Lats tumor suppressor kinase leads to cytoplasmic translocation and inactivation of the YAP oncoprotein. Furthermore, attenuation of this phosphorylation of YAP or Yorkie (Yki), the Drosophila homolog of YAP, potentiates their growth-promoting function in vivo. Moreover, YAP overexpression regulates gene expression in a manner opposite to cell density, and is able to overcome cell contact inhibition. Inhibition of YAP function restores contact inhibition in a human cancer cell line bearing deletion of Salvador (Sav), a Hippo pathway component. Interestingly, we observed that YAP protein is elevated and nuclear localized in some human liver and prostate cancers. Our observations demonstrate that YAP plays a key role in the Hippo pathway to control cell proliferation in response to cell contact.


Assuntos
Proliferação de Células , Inibição de Contato/genética , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Transativadores/antagonistas & inibidores , Transativadores/fisiologia , Proteínas 14-3-3/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Comunicação Celular/genética , Contagem de Células , Células Cultivadas , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Biológicos , Células NIH 3T3 , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Proteínas Quinases/metabolismo , Transporte Proteico , Transdução de Sinais/fisiologia , Transativadores/química , Transativadores/metabolismo , Proteínas de Sinalização YAP
18.
Curr Biol ; 16(21): 2090-100, 2006 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-16996265

RESUMO

BACKGROUND: The Hippo tumor-suppressor pathway has emerged as a key signaling pathway that controls tissue size in Drosophila. Merlin, the Drosophila homolog of the human Neurofibromatosis type-2 (NF2) tumor-suppressor gene, and the related protein Expanded are the most upstream components of the Hippo pathway identified so far. However, components acting upstream of Expanded and Merlin, such as transmembrane receptors, have not yet been identified. RESULTS: Here, we report that the protocadherin Fat acts as an upstream component in the Hippo pathway. Fat is a known tumor-suppressor gene in Drosophila, and fat mutants have severely overgrown imaginal discs. We found that the overgrowth phenotypes of fat mutants are similar to those of mutants in Hippo pathway components: fat mutant cells continued to proliferate after wild-type cells stopped proliferating, and fat mutant cells deregulated Hippo target genes such as cyclin E and diap1. Fat acts genetically and biochemically upstream of other Hippo pathway components such as Expanded, the Hippo and Warts kinases, and the transcriptional coactivator Yorkie. Fat is required for the stability of Expanded and its localization to the plasma membrane. In contrast, Fat is not required for Merlin localization, and Fat and Merlin act in parallel in growth regulation. CONCLUSIONS: Taken together, our data identify a cell-surface molecule that may act as a receptor of the Hippo signaling pathway.


Assuntos
Moléculas de Adesão Celular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Transdução de Sinais , Animais , Caderinas/genética , Caderinas/fisiologia , Moléculas de Adesão Celular/genética , Proliferação de Células , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Olho/embriologia , Olho/ultraestrutura , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Neurofibromina 2/metabolismo , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/metabolismo , Asas de Animais/anatomia & histologia , Proteínas de Sinalização YAP
19.
Biochemistry ; 44(19): 7305-14, 2005 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-15882069

RESUMO

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.


Assuntos
Cálcio/química , Motivos EF Hand/genética , Fatores de Crescimento Neural/química , Fragmentos de Peptídeos/química , Proteínas S100/química , Proteína Supressora de Tumor p53/química , Proteínas Supressoras de Tumor/química , Alanina/genética , Sequência de Aminoácidos , Animais , Ácido Glutâmico/genética , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Ratos , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/genética , Proteínas S100/metabolismo , Térbio/química , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
20.
J Med Chem ; 47(21): 5085-93, 2004 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-15456252

RESUMO

The binding of S100B to p53 down-regulates wild-type p53 tumor suppressor activity in cancer cells such as malignant melanoma, so a search for small molecules that bind S100B and prevent S100B-p53 complex formation was undertaken. Chemical databases were computationally searched for potential inhibitors of S100B, and 60 compounds were selected for testing on the basis of energy scoring, commercial availability, and chemical similarity clustering. Seven of these compounds bound to S100B as determined by steady state fluorescence spectroscopy (1.0 microM < or = K(D) < or = 120 microM) and five inhibited the growth of primary malignant melanoma cells (C8146A) at comparable concentrations (1.0 microM < or = IC(50) < or = 50 microM). Additionally, saturation transfer difference (STD) NMR experiments confirmed binding and qualitatively identified protons from the small molecule at the small molecule-S100B interface. Heteronuclear single quantum coherence (HSQC) NMR titrations indicate that these compounds interact with the p53 binding site on S100B. An NMR-docked model of one such inhibitor, pentamidine, bound to Ca(2+)-loaded S100B was calculated using intermolecular NOE data between S100B and the drug, and indicates that pentamidine binds into the p53 binding site on S100B defined by helices 3 and 4 and loop 2 (termed the hinge region).


Assuntos
Antineoplásicos/química , Cálcio/fisiologia , Fatores de Crescimento Neural/química , Proteínas S100/química , Proteína Supressora de Tumor p53/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Bases de Dados Factuais , Holoenzimas/química , Humanos , Espectroscopia de Ressonância Magnética , Melanoma , Modelos Moleculares , Conformação Molecular , Fatores de Crescimento Neural/metabolismo , Pentamidina/química , Ligação Proteica , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Espectrometria de Fluorescência , Proteína Supressora de Tumor p53/metabolismo
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