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1.
Nat Commun ; 15(1): 3343, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38637580

RESUMO

Pathogenic gut microbiota is responsible for a few debilitating gastrointestinal diseases. While the host immune cells do produce extracellular vesicles to counteract some deleterious effects of the microbiota, the extracellular vesicles are of insufficient doses and at unreliable exposure times. Here we use mechanical stimulation of hydrogel-embedded macrophage in a bioelectronic controller that on demand boost production of up to 20 times of therapeutic extracellular vesicles to ameliorate the microbes' deleterious effects in vivo. Our miniaturized wireless bioelectronic system termed inducible mechanical activation for in-situ and sustainable generating extracellular vesicles (iMASSAGE), leverages on wireless electronics and responsive hydrogel to impose mechanical forces on macrophages to produce extracellular vesicles that rectify gut microbiome dysbiosis and ameliorate colitis. This in vivo controllable extracellular vesicles-produced system holds promise as platform to treat various other diseases.


Assuntos
Colite , Vesículas Extracelulares , Microbioma Gastrointestinal , Microbiota , Humanos , Microbioma Gastrointestinal/fisiologia , Hidrogéis/farmacologia , Disbiose
2.
ACS Nano ; 18(13): 9613-9626, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38502546

RESUMO

Recent discoveries in commensal microbiota demonstrate the great promise of intratumoral bacteria as attractive molecular targets of tumors in improving cancer treatment. However, direct leveraging of in vivo antibacterial strategies such as antibiotics to potentiate cancer therapy often leads to uncertain effectiveness, mainly due to poor selectivity and potential adverse effects. Here, building from the clinical discovery that patients with breast cancer featured rich commensal bacteria, we developed an activatable biointerface by encapsulating commensal bacteria-derived extracellular vesicles (BEV) with a responsive nanocloak to potentiate immunoreactivity against intratumoral bacteria and breast cancer. We show that the interfacially cloaked BEV (cBEV) not only overcame serious systemic side responses but also demonstrated heightened immunogenicity by intercellular responsive immunogenicity, facilitating dendritic cell maturation through activating the cGAS-STING pathway. As a preventive measure, vaccination with nanocloaked cBEVs achieved strong protection against bacterial infection, largely providing prophylactic efficiency against tumor challenges. When treated in conjunction with immune checkpoint inhibitor anti-PD-L1 antibodies, the combined approach elicited a potent tumor-specific immune response, synergistically inhibiting tumor progression and mitigating lung metastases.


Assuntos
Neoplasias da Mama , Neoplasias , Humanos , Feminino , Imunoterapia , Neoplasias/terapia , Neoplasias da Mama/metabolismo , Imunidade , Bactérias , Microambiente Tumoral
3.
J Nanobiotechnology ; 21(1): 366, 2023 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-37798669

RESUMO

Extracellular vesicles (EVs) are membrane nanoarchitectures generated by cells that carry a variety of biomolecules, including DNA, RNA, proteins and metabolites. These characteristics make them attractive as circulating bioinformatic nanocabinets for liquid biopsy. Recent advances on EV biology and biogenesis demonstrate that EVs serve as highly important cellular surrogates involved in a wide range of diseases, opening up new frontiers for modern diagnostics. However, inefficient methods for EV enrichment, as well as low sensitivity of EV bioinformatic decoding technologies, hinder the use of EV nanocabinet for clinical diagnosis. To overcome these challenges, new EV nanotechnology is being actively developed to promote the clinical translation of EV diagnostics. This article aims to present the emerging enrichment strategies and bioinformatic decoding platforms for EV analysis, and their applications as bioinformatic nanomaterials in clinical settings.


Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Biópsia Líquida/métodos , Nanotecnologia , Biologia Computacional
4.
Sci Adv ; 9(25): eadg8719, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37352357

RESUMO

Animals evolved two defense strategies to survive infections. Antagonistic strategies include immune resistance mechanisms that operate to kill invading pathogens. Cooperative or physiological defenses mediate host adaptation to the infected state, limiting physiological damage and disease, without killing the pathogen, and have been shown to cause asymptomatic carriage and transmission of lethal pathogens. Here, we demonstrate that physiological defenses cooperate with the adaptive immune response to generate long-term asymptomatic carriage of the lethal enteric murine pathogen, Citrobacter rodentium. Asymptomatic carriage of genetically virulent C. rodentium provided immune resistance against subsequent infections. Immune protection was dependent on systemic antibody responses and pathogen virulence behavior rather than the recognition of specific virulent antigens. Last, we demonstrate that an avirulent strain of C. rodentium in the field has background mutations in genes that are important for LPS structure. Our work reveals insight into how asymptomatic infections can arise mechanistically with immune resistance, mediating exclusion of phenotypically virulent enteric pathogen to promote asymptomatic carriage.


Assuntos
Infecções por Enterobacteriaceae , Animais , Camundongos , Virulência , Intestino Delgado
5.
bioRxiv ; 2023 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-36711884

RESUMO

Animals have evolved two defense strategies to survive infections. Antagonistic strategies include mechanisms of immune resistance that operate to sense and kill invading pathogens. Cooperative or physiological defenses mediate host adaptation to the infected state, limiting physiological damage and disease, without killing the pathogen, and have been shown to cause asymptomatic carriage and transmission of lethal pathogens. Here we demonstrate that physiological defenses cooperate with the adaptive immune response to generate long-term asymptomatic carriage of the lethal enteric murine pathogen, Citrobacter rodentium. Asymptomatic carriage of genetically virulent C. rodentium provided immune resistance against subsequent infections. Host immune protection was dependent on systemic antibody responses and pathogen virulence behavior, rather than the recognition of specific virulent factor antigens. Finally, we demonstrate that an avirulent strain of C. rodentium in the field has background mutations in two genes that are important for LPS structure. Our work reveals novel insight into how asymptomatic infections can arise mechanistically with immune resistance, mediating exclusion of phenotypically virulent enteric pathogen to promote asymptomatic carriage.

6.
Nat Cell Biol ; 25(1): 159-169, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36635501

RESUMO

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the regulators of RAS signalling is incomplete. Here to uncover the impact of diverse KRAS-interacting proteins on lung cancer growth, we combined multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumour barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung cancer models, we show that HRAS and NRAS are suppressors of KRASG12D-driven tumour growth in vivo and confirm these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogues interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signalling. Furthermore, HRAS and NRAS mutations identified in oncogenic KRAS-driven human tumours partially abolished this effect. By comparing the tumour-suppressive effects of HRAS and NRAS in oncogenic KRAS- and oncogenic BRAF-driven lung cancer models, we confirm that RAS paralogues are specific suppressors of KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role RAS paralogue imbalance in oncogenic KRAS-driven lung cancer.


Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Camundongos , Animais , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transformação Celular Neoplásica/metabolismo , Transdução de Sinais/genética , Neoplasias Pulmonares/genética , Genes ras , Mutação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo
7.
J Cancer ; 12(11): 3257-3264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33976735

RESUMO

Purpose: Tumor blood vessels exhibit morphological and functional aberrancies. Its maturity and functionality are closely associated with colon cancer progression and therapeutic efficacy. The direct evidence proving whether oridonin (ORI) has vascular normalization promoting effect from which combination therapies will benefit is still lacking. Methods: We established a subcutaneous xenograft model of human colon cancer. The animals were divided into the Control and ORI-treated groups. Immunohistochemical analysis and TUNEL staining was applied to evaluate the proliferation, apoptosis and angiogenesis. Western blot analysis was employed to characterize the angiogenesis-related factors and JAK2/STAT3 signaling. Then, vascular normalization and macrophage reprogramming were assessed by immunofluorescence analysis. Results: The results showed that ORI obviously reduced tumor growth, diminished the numbers of Ki67+ cells and CD31+ microvessel density, while increased the numbers of TUNEL+ cells. The expression levels of VEGF and bFGF proteins were dramatically down-regulated while the angiostatin and endostatin levels were increased in the ORI-treated group. Moreover, ORI therapy remarkably promoted the pericyte coverage of tumor vessels from days 5 to 10, with the highest pericyte coverage rate occurred at day 7. In the time window of vascular normalization, hypoxia of the tumor microenvironment was improved by ORI, the expression of HIF-1a was downregulated. Moreover, CD206+ macrophage cells were diminished in the ORI-treated group. These anticancer effects of ORI maybe partly mediated by suppressing JAK2/STAT3 signaling pathway. Conclusions: These results highlight the potential effect of ORI on anti-angiogenesis and inducing vessel normalization roles of ORI, and probably provide optimum time point for the ORI therapy in conjunction with the chemoradiotherapy or immunotherapy.

8.
J Toxicol Pathol ; 34(1): 95-99, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33627949

RESUMO

Glioblastoma (GBM) is a highly aggressive central nervous system cancer. Its extracranial metastases have rarely been reported in the past few decades. Moreover, the pathogenesis of extracranial GBM metastases remains unclear. Here, we report a case of pulmonary metastasis in a male Wistar rat of C6 GBM model. This reported Wistar male rat was one of the experimental control group without any other intervention except for C6 GBM cells orthotopic implantation. On postoperative day 15, the animal which was reported in this study showed highly cellular, pleomorphic, tumor with nuclear atypia in the brain (Ki67, approximately 65.7%) and lungs (Ki67, 49.5%). Tumor cells in the lung showed immunoreactivity for glial fibrillary acidic protein. Inflammatory CD68+ cell infiltration, weakly positive E-cadherin, and strongly positive staining for vimentin were observed both in tumors in the brain and lungs. Based on further morphological analysis, we speculate that the potential metastatic route into the lung might be hematogenous metastasis.

9.
Proc Natl Acad Sci U S A ; 117(22): 12121-12130, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32424096

RESUMO

HRAS, NRAS, and KRAS4A/KRAS4B comprise the RAS family of small GTPases that regulate signaling pathways controlling cell proliferation, differentiation, and survival. RAS pathway abnormalities cause developmental disorders and cancers. We found that KRAS4B colocalizes on the cell membrane with other RAS isoforms and a subset of prenylated small GTPase family members using a live-cell quantitative split luciferase complementation assay. RAS protein coclustering is mainly mediated by membrane association-facilitated interactions (MAFIs). Using the RAS-RBD (CRAF RAS binding domain) interaction as a model system, we showed that MAFI alone is not sufficient to induce RBD-mediated RAS inhibition. Surprisingly, we discovered that high-affinity membrane-targeted RAS binding proteins inhibit RAS activity and deplete RAS proteins through an autophagosome-lysosome-mediated degradation pathway. Our results provide a mechanism for regulating RAS activity and protein levels, a more detailed understanding of which should lead to therapeutic strategies for inhibiting and depleting oncogenic RAS proteins.


Assuntos
Autofagossomos/metabolismo , Membrana Celular/metabolismo , Lisossomos/metabolismo , Proteínas ras/metabolismo , Humanos , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Transdução de Sinais
10.
Cell Rep ; 29(11): 3448-3459.e6, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31825828

RESUMO

Oncogenic RAS mutations drive cancers at many sites. Recent reports suggest that RAS dimerization, multimerization, and clustering correlate strongly with activation of RAS signaling. We have found that re-expression of DIRAS3, a RAS-related small GTPase tumor suppressor that is downregulated in multiple cancers, inhibits RAS/mitogen-activated protein kinase (MAPK) signaling by interacting directly with RAS-forming heteromers, disrupting RAS clustering, inhibiting Raf kinase activation, and inhibiting transformation and growth of cancer cells and xenografts. Disruption of K-RAS cluster formation requires the N terminus of DIRAS3 and interaction of both DIRAS3 and K-RAS with the plasma membrane. Interaction of DIRAS3 with both K-RAS and H-RAS suggests a strategy for inhibiting oncogenic RAS function.


Assuntos
Carcinogênese/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas rho de Ligação ao GTP/metabolismo , Células 3T3 , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/metabolismo
11.
Opt Express ; 25(21): 26160-26165, 2017 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-29041276

RESUMO

A flexible chalcogenide fiber bundle (FB) with a resolution as high as ~31 lp/mm has been fabricated for delivering thermal images of objects at room temperature. The FB is composed of ~200,000 single fibers with a Ge-As-Te-Se glass core 15 µm in diameter and a polyetherimide (PEI) cladding 16.8 µm in diameter. These Ge-As-Te-Se/PEI fibers show good transparency in the 3-12 µm spectral region. The fabricated FB presents a filling factor of ~72% and a crosstalk of ~1%. High-quality thermal images of a human hand were obtained through the FB, demonstrating good potential of the FB for longwave infrared imaging in the areas such as medicine, industry and defense.

12.
Oncotarget ; 7(13): 15986-6002, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26910119

RESUMO

BH3 mimetic compounds induce tumor cell death through targeted inhibition of anti-apoptotic BCL2 proteins. Resistance to one such compound, ABT-737, is due to increased levels of anti-apoptotic MCL1. Using chemical and genetic approaches, we show that resistance to ABT-737 is abrogated by inhibition of the mitochondrial RING E3 ligase, MARCH5. Mechanistically, this is due to increased expression of pro-apoptotic BCL2 family member, NOXA, and is associated with MARCH5 regulation of MCL1 ubiquitylation and stability in a NOXA-dependent manner. MARCH5 expression contributed to an 8-gene signature that correlates with sensitivity to the preclinical BH3 mimetic, navitoclax. Furthermore, we observed a synthetic lethal interaction between MCL1 and MARCH5 in MCL1-dependent breast cancer cells. Our data uncover a novel level at which the BCL2 family is regulated; furthermore, they suggest targeting MARCH5-dependent signaling will be an effective strategy for treatment of BH3 mimetic-resistant tumors, even in the presence of high MCL1.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas de Membrana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Compostos de Anilina/farmacologia , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/efeitos dos fármacos , Nitrofenóis/farmacologia , Fragmentos de Peptídeos , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Sulfonamidas/farmacologia , Ubiquitina-Proteína Ligases/efeitos dos fármacos
13.
Opt Express ; 24(26): 30031-30037, 2016 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-28059388

RESUMO

Using a home-made black phosphorus plate (BPP) as handedness controller and Q-switch modulator synchronously, a ~1.6 µm pulsed vortex laser with well-determined handedness is demonstrated in this letter. Stable vortex pulses of LG0, + 1, LG0,-1, LG0, + 2 and LG0,-2 modes were respectively achieved from compact resonant cavities in this experiment. Such pulsed vortex laser should have promising applications in various fields based on its simple structure, controllable handedness, and carried orbital angular momentum.

14.
Biochim Biophys Acta ; 1863(2): 284-92, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26646257

RESUMO

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways.


Assuntos
Proteínas de Artrópodes/metabolismo , Proteínas de Insetos/metabolismo , Luciferases/metabolismo , Mapeamento de Interação de Proteínas/métodos , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Western Blotting , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Decápodes/enzimologia , Decápodes/genética , Vaga-Lumes/enzimologia , Vaga-Lumes/genética , Genes Reporter/genética , Humanos , Proteínas de Insetos/genética , Luciferases/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Cell Rep ; 9(5): 1946-1958, 2014 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-25464845

RESUMO

Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Genes Reporter , Humanos , Luciferases de Vaga-Lume/biossíntese , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Recombinases/fisiologia , Proteína Supressora de Tumor p53/genética
16.
PLoS One ; 8(3): e58395, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23516471

RESUMO

Functional and mechanistic studies of Wnt signaling have been severely hindered by the inaccessibility of bioactive proteins. To overcome this long-standing barrier, we engineered and characterized a panel of Chinese hamster ovary (CHO) cell lines with inducible transgenes encoding tagged and un-tagged human WNT1, WNT3A, WNT5A, WNT7A, WNT11, WNT16 or the soluble Wnt antagonist Fzd8CRD, all integrated into an identical genomic locus. Using a quantitative real-time bioluminescence assay, we show that cells expressing WNT1, 3A and 7A stimulate Wnt/beta-catenin reporter activity, while the other WNT expressing cell lines interfere with this activation. Additionally, in contrast to WNT3A, WNT1 only exhibits activity when cell-associated, and thus only signals to neighboring cells. The reporter assay also revealed a rapid decline of Wnt activity at 37°C, indicating that Wnt activity is highly labile. These engineered cell lines will reduce the cost of making and purifying Wnt proteins and serve as a continuous, reliable and regulatable source of Wnts to research laboratories around the world.


Assuntos
Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Células CHO , Cricetinae , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Wnt/isolamento & purificação
17.
Nat Rev Cancer ; 13(2): 83-96, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23303139

RESUMO

The MDM2 and MDMX (also known as HDMX and MDM4) proteins are deregulated in many human cancers and exert their oncogenic activity predominantly by inhibiting the p53 tumour suppressor. However, the MDM proteins modulate and respond to many other signalling networks in which they are embedded. Recent mechanistic studies and animal models have demonstrated how functional interactions in these networks are crucial for maintaining normal tissue homeostasis, and for determining responses to oncogenic and therapeutic challenges. This Review highlights the progress made and pitfalls encountered as the field continues to search for MDM-targeted antitumour agents.


Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Transformação Celular Neoplásica/genética , Humanos , Neoplasias/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética
18.
Cancer Cell ; 21(5): 595-596, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22624708

RESUMO

In this issue of Cancer Cell, Gannon and colleagues create genetically engineered mice to test the role phosphorylation plays in the modification of one serine long thought to play a critical role in controlling the activity of MDM2, one of p53's main negative regulators.

19.
Mol Cell ; 31(5): 650-9, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18775325

RESUMO

The yeast Sir2/3/4 complex forms a heterochromatin-like structure that represses transcription. The proteins nucleate at silencers and spread distally, utilizing the Sir2 NAD(+)-dependent histone deacetylase activity and the affinity of Sir3/4 for deacetylated histone tails. A by-product of the Sir2 reaction, O-acetyl-ADP-ribose (OAADPr), is thought to aid spreading by binding one of the Sir proteins. We developed a protein chimera approach to reexamine the contributions of Sir2. We show that a Sir3 chimera-bearing Hos3, an unrelated NAD(+)-independent histone deacetylase, substitutes for Sir2 in silencing. Sir3-Hos3 operates within the Sir pathway, spreading while deacetylating histones. Moreover, the chimera represses HM loci in strains lacking all five OAADPr-producing deacetylases, indicating that OAADPr is not necessary for silencing. Repression by a Hos3 hybrid bearing the targeting motifs of Sir2 shows that targeting doesn't require the Sir2 reaction. Together, these data demonstrate that protein deacetylation is the only essential function of Sir2 in creating silenced chromatin.


Assuntos
Inativação Gênica , Histona Desacetilases/metabolismo , O-Acetil-ADP-Ribose/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuínas/metabolismo , Transcrição Gênica , Histona Desacetilases/genética , Modelos Moleculares , O-Acetil-ADP-Ribose/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2 , Sirtuínas/genética
20.
Proc Natl Acad Sci U S A ; 104(30): 12365-70, 2007 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-17640893

RESUMO

P53 regulates numerous downstream targets to induce cell cycle arrest, senescence, apoptosis, and DNA repair in response to diverse stresses. Hdm2 and Hdmx are critical negative regulators of P53 because Hdm2 regulates P53 abundance, and both can antagonize P53 transactivation. Modest changes in Hdm2 or Hdmx abundance affect P53 regulation, yet quantitative information regarding their endogenous intracellular concentrations and subcellular distributions during a stress response are lacking. We analyzed these parameters in normal and cancer cells after DNA damage. Our data show that the nuclear abundance of Hdm2 and Hdmx relative to P53 limits P53 activity in cells growing in culture. Upon DNA damage, P53 nuclear abundance increases, whereas Hdm2 and Hdmx stability decreases, which greatly limits their ability to antagonize P53, regardless of their levels. These data indicate that the damage-activated switch in Hdm2 ubiquitin ligase preference from P53 to itself and Hdmx is central to P53 activation.


Assuntos
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , DNA/genética , Dano ao DNA/genética , Regulação da Expressão Gênica , Humanos , Cinética , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética
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