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1.
Cell ; 163(6): 1457-67, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26627735

RESUMO

A variety of signals finely tune insulin secretion by pancreatic ß cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in ß cells is critical for insulin secretion. Mice lacking COP1 in ß cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human ß-cell pathophysiology. In normal ß cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/metabolismo , Exocitose , Deleção de Genes , Glucose/metabolismo , Humanos , Hiperglicemia/metabolismo , Secreção de Insulina , Camundongos , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética
2.
Nature ; 591(7848): 131-136, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472215

RESUMO

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Morte Celular , Membrana Celular/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Apoptose , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Morte Celular/genética , Feminino , Humanos , Macrófagos , Masculino , Camundongos , Mutação , Necrose , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Multimerização Proteica , Piroptose/genética
3.
Nature ; 575(7784): 679-682, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723262

RESUMO

Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR11, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL2-4. Mice that lack caspase-8 display MLKL-dependent embryonic lethality4, as do mice that express catalytically inactive CASP8(C362A)5. Casp8C362A/C362AMlkl-/- mice die during the perinatal period5, whereas Casp8-/-Mlkl-/- mice are viable4, which indicates that inactive caspase-8 also has a pro-death scaffolding function. Here we show that mutant CASP8(C362A) induces the formation of ASC (also known as PYCARD) specks, and caspase-1-dependent cleavage of GSDMD and caspases 3 and 7 in MLKL-deficient mouse intestines around embryonic day 18. Caspase-1 and its adaptor ASC contributed to the perinatal lethal phenotype because a number of Casp8C362A/C362AMlkl-/-Casp1-/- and Casp8C362A/C362AMlkl-/-Asc-/- mice survived beyond weaning. Transfection studies suggest that inactive caspase-8 adopts a distinct conformation to active caspase-8, enabling its prodomain to engage ASC. Upregulation of the lipopolysaccharide sensor caspase-11 in the intestines of both Casp8C362A/C362AMlkl-/- and Casp8C362A/C362AMlkl-/-Casp1-/- mice also contributed to lethality because Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- (Casp11 is also known as Casp4) neonates survived more often than Casp8C362A/C362AMlkl-/-Casp1-/- neonates. Finally, Casp8C362A/C362ARipk3-/-Casp1-/-Casp11-/- mice survived longer than Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- mice, indicating that a necroptosis-independent function of RIPK3 also contributes to lethality. Thus, unanticipated plasticity in death pathways is revealed when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.


Assuntos
Caspase 8/metabolismo , Morte Celular/genética , Mucosa Intestinal/citologia , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 8/genética , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Mucosa Intestinal/enzimologia , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
4.
Proc Natl Acad Sci U S A ; 119(38): e2123117119, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36099298

RESUMO

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.


Assuntos
Acinetobacter baumannii , Antibacterianos , Ácido Aspártico Endopeptidases , Proteínas de Bactérias , Farmacorresistência Bacteriana , Furanos , Deleção de Genes , Lipoproteínas , Inibidores de Proteases , Piridinas , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Ácido Aspártico Endopeptidases/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Furanos/farmacologia , Lipoproteínas/biossíntese , Lipoproteínas/genética , Peptídeos/farmacologia , Inibidores de Proteases/farmacologia , Sinais Direcionadores de Proteínas/genética , Piridinas/farmacologia
5.
J Immunol ; 202(1): 183-193, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30510070

RESUMO

Both common and rare genetic variants of laccase domain-containing 1 (LACC1, previously C13orf31) are associated with inflammatory bowel disease, leprosy, Behcet disease, and systemic juvenile idiopathic arthritis. However, the functional relevance of these variants is unclear. In this study, we use LACC1-deficient mice to gain insight into the role of LACC1 in regulating inflammation. Following oral administration of Citrobacter rodentium, LACC1 knockout (KO) mice had more severe colon lesions compared with wildtype (WT) controls. Immunization with collagen II, a collagen-induced arthritis (CIA) model, resulted in an accelerated onset of arthritis and significantly worse arthritis and inflammation in LACC1 KO mice. Similar results were obtained in a mannan-induced arthritis model. Serum and local TNF in CIA paws and C. rodentium colons were significantly increased in LACC1 KO mice compared with WT controls. The percentage of IL-17A-producing CD4+ T cells was elevated in LACC1 KO mice undergoing CIA as well as aged mice compared with WT controls. Neutralization of IL-17, but not TNF, prevented enhanced mannan-induced arthritis in LACC1 KO mice. These data provide new mechanistic insight into the function of LACC1 in regulating TNF and IL-17 during inflammatory responses. We hypothesize that these effects contribute to immune-driven pathologies observed in individuals carrying LACC1 variants.


Assuntos
Artrite Experimental/imunologia , Artrite Juvenil/imunologia , Citrobacter rodentium/fisiologia , Infecções por Enterobacteriaceae/imunologia , Doenças Inflamatórias Intestinais/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxirredutases/metabolismo , Células Th17/imunologia , Alelos , Animais , Artrite Experimental/microbiologia , Artrite Juvenil/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Doenças Inflamatórias Intestinais/genética , Interleucina-17/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases/genética , Polimorfismo Genético , Fatores de Necrose Tumoral/metabolismo
6.
Proc Natl Acad Sci U S A ; 112(18): 5679-84, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25902490

RESUMO

TNF superfamily death ligands are expressed on the surface of immune cells and can trigger apoptosis in susceptible cancer cells by engaging cognate death receptors. A recombinant soluble protein comprising the ectodomain of Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has shown remarkable preclinical anticancer activity but lacked broad efficacy in patients, possibly owing to insufficient exposure or potency. We observed that antibody cross-linking substantially enhanced cytotoxicity of soluble Apo2L/TRAIL against diverse cancer cell lines. Presentation of the ligand on glass-supported lipid bilayers enhanced its ability to drive receptor microclustering and apoptotic signaling. Furthermore, covalent surface attachment of Apo2L/TRAIL onto liposomes--synthetic lipid-bilayer nanospheres--similarly augmented activity. In vivo, liposome-displayed Apo2L/TRAIL achieved markedly better exposure and antitumor activity. Thus, covalent synthetic-membrane attachment of a cell-surface ligand enhances efficacy, increasing therapeutic potential. These findings have translational implications for liposomal approaches as well as for Apo2L/TRAIL and other clinically relevant TNF ligands.


Assuntos
Antineoplásicos/química , Membrana Celular/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose , Biotinilação , Ligante CD27/metabolismo , Caspase 8/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Epitopos/química , Proteína Ligante Fas/metabolismo , Humanos , Sistema Imunitário , Imunoterapia/métodos , Concentração Inibidora 50 , Ligantes , Lipossomos/química , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Transplante de Neoplasias , Neoplasias/imunologia , Neoplasias/metabolismo , Proteínas Recombinantes/metabolismo
9.
Breast Cancer Res ; 17: 59, 2015 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-25902869

RESUMO

INTRODUCTION: Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis. METHODS: PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting. RESULTS: We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-4) and P=0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis. CONCLUSIONS: Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Microtúbulos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Moduladores de Tubulina/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Docetaxel , Sinergismo Farmacológico , Feminino , Amplificação de Genes , Expressão Gênica , Humanos , Prognóstico , Transdução de Sinais/efeitos dos fármacos , Taxoides/farmacologia , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo
10.
Blood ; 122(22): 3678-90, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-23886837

RESUMO

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.


Assuntos
Proteínas de Transporte/fisiologia , Endotélio Vascular/embriologia , Endotélio Vascular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Junções Intercelulares/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neovascularização Fisiológica , Gravidez , Interferência de RNA , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia
11.
Proc Natl Acad Sci U S A ; 109(47): 19368-73, 2012 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-23134728

RESUMO

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.


Assuntos
Neoplasias/enzimologia , Neoplasias/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Células NIH 3T3 , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
12.
PLoS One ; 18(4): e0280484, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37079518

RESUMO

BACKGROUND: The basis of Age-related macular degeneration (AMD) genetic risk has been well documented; however, few studies have looked at genetic biomarkers of disease progression or treatment response within advanced AMD patients. Here we report the first genome-wide analysis of genetic determinants of low-luminance vision deficit (LLD), which is seen as predictive of visual acuity loss and anti-VEGF treatment response in neovascular AMD patients. METHODS: AMD patients were separated into small- and large-LLD groups for comparison and whole genome sequencing was performed. Genetic determinants of LLD were assessed by common and rare variant genetic analysis. Follow-up functional analysis of rare coding variants identified by the burden test was then performed in vitro. RESULTS: We identified four coding variants in the CIDEC gene. These rare variants were only present in patients with a small LLD, which has been previously shown to indicate better prognosis and better anti-VEGF treatment response. Our in vitro functional characterization of these CIDEC alleles revealed that all decrease the binding affinity between CIDEC and the lipid droplet fusion effectors PLIN1, RAB8A and AS160. The rare CIDEC alleles all cause a hypomorphic defect in lipid droplet fusion and enlargement, resulting in a decreased fat storage capability in adipocytes. CONCLUSIONS: As we did not detect CIDEC expression in the ocular tissue affected by AMD, our results suggest that the CIDEC variants do not play a direct role in the eye and influence low-luminance vision deficit via an indirect and systemic effect related to fat storage capacity.


Assuntos
Baixa Visão , Degeneração Macular Exsudativa , Humanos , Inibidores da Angiogênese , Gotículas Lipídicas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Acuidade Visual/genética , Degeneração Macular Exsudativa/metabolismo
13.
Mol Ther Nucleic Acids ; 32: 773-793, 2023 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-37346977

RESUMO

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery in vivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.

14.
Cell Metab ; 34(9): 1377-1393.e8, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-35987202

RESUMO

Fibrosis is the major risk factor associated with morbidity and mortality in patients with non-alcoholic steatohepatitis (NASH)-driven chronic liver disease. Although numerous efforts have been made to identify the mediators of the initiation of liver fibrosis, the molecular underpinnings of fibrosis progression remain poorly understood, and therapies to arrest liver fibrosis progression are elusive. Here, we identify a pathway involving WNT1-inducible signaling pathway protein 1 (WISP1) and myocardin-related transcription factor (MRTF) as a central mechanism driving liver fibrosis progression through the integrin-dependent transcriptional reprogramming of myofibroblast cytoskeleton and motility. In mice, WISP1 deficiency protects against fibrosis progression, but not fibrosis onset. Moreover, the therapeutic administration of a novel antibody blocking WISP1 halted the progression of existing liver fibrosis in NASH models. These findings implicate the WISP1-MRTF axis as a crucial determinant of liver fibrosis progression and support targeting this pathway by antibody-based therapy for the treatment of NASH fibrosis.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Fatores de Transcrição , Animais , Fígado/metabolismo , Cirrose Hepática/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Nucleares , Transdução de Sinais , Transativadores , Fatores de Transcrição/metabolismo
15.
Adv Genet (Hoboken) ; 2(1): e10036, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36618440

RESUMO

ERBB3 is a pseudokinase domain-containing member of the ERBB family of receptor tyrosine kinases (RTKs). Following ligand binding, ERBB receptors homo- or hetero-dimerize, leading to a head-to-tail arrangement of the intracellular kinase domains, where the "receiver" kinase domain of one ERBB is activated by the "activator" domain of the other ERBB in the dimer. In ERBB3, a conserved valine at codon 943 (V943) in the kinase C-terminal domain has been shown to be important for its function as an "activator" kinase in vitro. Here we report a knock-in mouse model where we have modified the endogenous Erbb3 allele to allow for tissue-specific conditional expression of Erbb3 V943R (Erbb3 CKI-V943R ). Additionally, we generated an Erbb3 D850N (Erbb3 CKI-D850N ) conditional knock-in mouse model where the conserved aspartate in the DFG motif of the pseudokinase domain was mutated to abolish any potential residual kinase activity. While Erbb3 D850N/D850N animals developed normally, homozygous Erbb3 V943R/V943R expression during development resulted in embryonic lethality. Further, tissue specific expression of Erbb3 V943R/V943R in the mammary gland epithelium following its activation using MMTV-Cre resulted in delayed elongation of the ductal network during puberty. Single-cell RNA-seq analysis of Erbb3 V943R/V943R mammary glands showed a reduction in a specific subset of fibrinogen-producing luminal epithelial cells.

16.
J Histochem Cytochem ; 68(1): 9-23, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31385742

RESUMO

Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workflow takes advantage of the very large scan fields possible with modern FE-SEMs that allow for the acquisition of wide-field overview images which can be explored at the ultrastructural level by digitally zooming into the images. Examples from applications in pulmonary research and neuropathology demonstrate the versatility and efficiency of this method. This BSE-SEM-based multiscale imaging procedure promises to substantially simplify and accelerate ultrastructural tissue analysis in pathology.


Assuntos
Microscopia Eletrônica de Varredura , Patologia/métodos , Animais , Glomérulos Renais/diagnóstico por imagem , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Pulmão/diagnóstico por imagem , Pulmão/patologia , Camundongos , Placa Amiloide/diagnóstico por imagem , Placa Amiloide/patologia , Placa Amiloide/ultraestrutura , Ratos
17.
mBio ; 11(5)2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900806

RESUMO

Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use.IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.


Assuntos
Antibacterianos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Farmacorresistência Bacteriana/genética , Lipoproteínas/metabolismo , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/enzimologia , Animais , Ácido Aspártico Endopeptidases/genética , Proteínas de Bactérias/genética , Enterobacteriaceae/classificação , Enterobacteriaceae/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/farmacologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade
18.
Nat Genet ; 52(1): 106-117, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31907489

RESUMO

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the 'venom-ome' and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 'venom-ome-specific toxins' (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.


Assuntos
Biologia Computacional/métodos , Venenos Elapídicos/análise , Venenos Elapídicos/genética , Genoma , Naja naja/genética , Transcriptoma , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Índia , Homologia de Sequência
19.
Eukaryot Cell ; 6(12): 2354-64, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17766466

RESUMO

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.


Assuntos
Flagelos/metabolismo , Regulação da Expressão Gênica , Interfase , Cinesinas/fisiologia , Microtúbulos/metabolismo , Mitose , Animais , Giardia lamblia , Proteínas de Fluorescência Verde/metabolismo , Cinesinas/metabolismo , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/química , Modelos Biológicos , Nocodazol/farmacologia , Paclitaxel/farmacologia , Moduladores de Tubulina/farmacologia
20.
Nat Commun ; 8(1): 862, 2017 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-29021563

RESUMO

Members of the ISWI family of chromatin remodelers mobilize nucleosomes to control DNA accessibility and, in some cases, are required for recovery from DNA damage. However, it remains poorly understood how the non-catalytic ISWI subunits BAZ1A and BAZ1B might contact chromatin to direct the ATPase SMARCA5. Here, we find that the plant homeodomain of BAZ1A, but not that of BAZ1B, has the unusual function of binding DNA. Furthermore, the BAZ1A bromodomain has a non-canonical gatekeeper residue and binds relatively weakly to acetylated histone peptides. Using CRISPR-Cas9-mediated genome editing we find that BAZ1A and BAZ1B each recruit SMARCA5 to sites of damaged chromatin and promote survival. Genetic engineering of structure-designed bromodomain and plant homeodomain mutants reveals that reader modules of BAZ1A and BAZ1B, even when non-standard, are critical for DNA damage recovery in part by regulating ISWI factors loading at DNA lesions and supporting transcriptional programs required for survival.ISWI chromatin remodelers regulate DNA accessibility and have been implicated in DNA damage repair. Here, the authors uncover functions, in response to DNA damage, for the bromodomain of the ISWI subunit BAZ1B and for the non-canonical PHD and bromodomain modules of the paralog BAZ1A.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Fatores de Transcrição/fisiologia , Sistemas CRISPR-Cas , Linhagem Celular , Cromatina/metabolismo , DNA/metabolismo , Edição de Genes , Humanos , Estrutura Molecular , Fatores de Transcrição/química
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