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1.
FEBS Open Bio ; 2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34431239

RESUMO

Expanding on previous demonstrations of the therapeutic effects of adeno-associated virus (AAV) carrying small-hairpin RNA (shRNA) in downregulating the mechanistic target of rapamycin (mTOR) in in vivo retinal vascular disorders, vascular endothelial growth factor (VEGF)-stimulated endothelial cells were treated with AAV2-shmTOR to examine the role of mTOR inhibition in retinal angiogenesis. AAV2-shmTOR exposure significantly reduced mTOR expression in human umbilical vein endothelial cells (HUVECs) and decreased downstream signaling cascades of mTOR complex 1 (mTORC1) and mTORC2 under VEGF treatment. Moreover, the angiogenic potential of VEGF was significantly inhibited by AAV2-shmTOR, which preserved endothelial integrity by maintaining tight junctions between HUVECs. These data thus support previous in vivo studies and provide evidence that AAV2-shmTOR induces therapeutic effects by inhibiting the neovascularization of endothelial cells.

2.
Nat Commun ; 12(1): 3338, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099686

RESUMO

The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.


Assuntos
Microscopia Crioeletrônica , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIH/química , Adutos de DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIH/genética
3.
Nat Commun ; 12(1): 3487, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108468

RESUMO

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.


Assuntos
Alquil e Aril Transferases/química , Diterpenos/metabolismo , Proteínas Fúngicas/química , Alquil e Aril Transferases/metabolismo , Ascomicetos/química , Ascomicetos/enzimologia , Catálise , Domínio Catalítico , Microscopia Crioeletrônica , Ciclização , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeos/biossíntese , Liases/química , Liases/metabolismo , Enzimas Multifuncionais , Fosfatos de Poli-Isoprenil/metabolismo , Conformação Proteica
4.
Sci Adv ; 7(15)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33827808

RESUMO

During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5-Å resolution cryo-electron microscopy (cryo-EM) structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologs of CDK7, cyclin H, and MAT1, respectively. The carboxyl-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with the previous cryo-EM structure of the preinitiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK.

5.
Nat Commun ; 12(1): 929, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568648

RESUMO

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Rhodobacter capsulatus/enzimologia , Proteínas de Bactérias/genética , Domínio Catalítico , Coenzimas/química , Coenzimas/metabolismo , Microscopia Crioeletrônica , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Engenharia Genética , Rhodobacter capsulatus/química , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo
6.
Sci Adv ; 7(3)2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33523904

RESUMO

The Cdk8 kinase module (CKM) in Mediator, comprising Med13, Med12, CycC, and Cdk8, regulates RNA polymerase II transcription through kinase-dependent and -independent functions. Numerous pathogenic mutations causative for neurodevelopmental disorders and cancer congregate in CKM subunits. However, the structure of the intact CKM and the mechanism by which Cdk8 is non-canonically activated and functionally affected by oncogenic CKM alterations are poorly understood. Here, we report a cryo-electron microscopy structure of Saccharomyces cerevisiae CKM that redefines prior CKM structural models and explains the mechanism of Med12-dependent Cdk8 activation. Med12 interacts extensively with CycC and activates Cdk8 by stabilizing its activation (T-)loop through conserved Med12 residues recurrently mutated in human tumors. Unexpectedly, Med13 has a characteristic Argonaute-like bi-lobal architecture. These findings not only provide a structural basis for understanding CKM function and pathological dysfunction, but also further impute a previously unknown regulatory mechanism of Mediator in transcriptional modulation through its Med13 Argonaute-like features.

7.
Invest Ophthalmol Vis Sci ; 61(2): 45, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32106292

RESUMO

Purpose: Recent studies have shown that inhibitors of the mechanistic target of rapamycin (mTOR) play important roles in proliferating endothelial cells within the retinal vasculature. Here we explore the effects of inhibiting mTOR as a potential gene therapeutic against pathological retinal angiogenesis in a rat model of oxygen-induced retinopathy (OIR). Methods: Sprague-Dawley pups were used to generate the OIR model, with a recombinant adeno-associated virus expressing an shRNA (rAAV2-shmTOR-GFP) being administered via intravitreal injection on returning the rats to normoxia, with appropriate controls. Immunohistochemistry and TUNEL assays, as well as fluorescein angiography, were performed on transverse retinal sections and flat mounts, respectively, to determine the in vivo effects of mTOR inhibition. Results: Compared with normal control rats, as well as OIR model animals that were either untreated (20.95 ± 6.85), mock-treated (14.50 ± 2.47), or injected with a control short hairpin RNA (shRNA)-containing virus vector (16.64 ± 4.92), rAAV2-shmTOR-GFP (4.28 ± 2.86, P = 0.00103) treatment resulted in dramatically reduced neovascularization as a percentage of total retinal area. These results mirrored quantifications of retinal avascular area and vessel tortuosity, with rAAV2-shmTOR-GFP exhibiting significantly greater therapeutic efficacy than the other treatments. The virus vector was additionally shown to reduce inflammatory cell infiltration into retinal tissue and possess antiapoptotic properties, both these processes having been implicated in the pathophysiology of angiogenic retinal disorders. Conclusions: Taken together, these results demonstrate the strong promise of rAAV2-shmTOR-GFP as an effective and convenient gene therapy for the treatment of neovascular retinal diseases.


Assuntos
Dependovirus/genética , Técnicas de Silenciamento de Genes/métodos , Terapia Genética/métodos , Neovascularização Retiniana/terapia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Interferência de RNA , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley
8.
G3 (Bethesda) ; 9(11): 3791-3800, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690598

RESUMO

A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.


Assuntos
Linhagem da Célula , Drosophila/genética , Animais , Encéfalo , Olho , Expressão Gênica , Sistema Linfático , Pesquisa , Estudantes , Universidades , Asas de Animais
9.
J Proteome Res ; 18(10): 3586-3596, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31498634

RESUMO

The enrichment of biotinylated proteins using immobilized streptavidin has become a staple methodology for affinity purification-based proteomics. Many of these workflows rely upon tryptic digestion to elute streptavidin-captured moieties from the beads. The concurrent release of high amounts of streptavidin-derived peptides into the digested sample, however, can significantly hamper the effectiveness of downstream proteomic analyses by increasing the complexity and dynamic range of the mixture. Here, we describe a strategy for the chemical derivatization of streptavidin that renders it largely resistant to proteolysis by trypsin and thereby dramatically reduces the amount of streptavidin contamination in the sample. This rapid and robust approach improves the effectiveness of mass spectrometry-based characterization of streptavidin-purified samples making it broadly useful for a wide variety of applications. In addition, we show that this chemical protection strategy can also be applied to other affinity matrices including immobilized antibodies against HA epitopes.


Assuntos
Proteólise , Estreptavidina/química , Tripsina/metabolismo , Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Proteômica/métodos
10.
Genome Biol ; 20(1): 181, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31464627

RESUMO

BACKGROUND: Birds of prey (raptors) are dominant apex predators in terrestrial communities, with hawks (Accipitriformes) and falcons (Falconiformes) hunting by day and owls (Strigiformes) hunting by night. RESULTS: Here, we report new genomes and transcriptomes for 20 species of birds, including 16 species of birds of prey, and high-quality reference genomes for the Eurasian eagle-owl (Bubo bubo), oriental scops owl (Otus sunia), eastern buzzard (Buteo japonicus), and common kestrel (Falco tinnunculus). Our extensive genomic analysis and comparisons with non-raptor genomes identify common molecular signatures that underpin anatomical structure and sensory, muscle, circulatory, and respiratory systems related to a predatory lifestyle. Compared with diurnal birds, owls exhibit striking adaptations to the nocturnal environment, including functional trade-offs in the sensory systems, such as loss of color vision genes and selection for enhancement of nocturnal vision and other sensory systems that are convergent with other nocturnal avian orders. Additionally, we find that a suite of genes associated with vision and circadian rhythm are differentially expressed in blood tissue between nocturnal and diurnal raptors, possibly indicating adaptive expression change during the transition to nocturnality. CONCLUSIONS: Overall, raptor genomes show genomic signatures associated with the origin and maintenance of several specialized physiological and morphological features essential to be apex predators.


Assuntos
Evolução Biológica , Ritmo Circadiano/genética , Genoma , Comportamento Predatório/fisiologia , Aves Predatórias/genética , Adaptação Fisiológica/genética , Animais , Filogenia
11.
Mol Ther Methods Clin Dev ; 14: 171-179, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31380463

RESUMO

Choroidal neovascularization (CNV) is the defining characteristic of the wet subtype of age-related macular degeneration (AMD), which is a rapidly growing global health problem. Previously, we had demonstrated the therapeutic potential of gene therapy against CNV using short hairpin RNA (shRNA) delivered via recombinant adeno-associated virus (rAAV), which abrogates mammalian-to-mechanistic (mTOR) activity in a novel manner by simultaneously inhibiting both mTOR complexes. Both the target and use of gene therapy represent a novel treatment modality against AMD. Here, the xenogeneic GFP gene used as a reporter in previous studies was removed from the virus vector to further develop the therapeutic for clinical trials. Instead, a stuffer DNA derived from the 3' UTR of the human UBE3A gene was used to ensure optimal viral genome size for efficient rAAV assembly. The virus vector containing the stuffer DNA, rAAV2-shmTOR-SD, positively compares to one encoding the shRNA and a GFP expression cassette in terms of reducing CNV in a laser-induced mouse model, as determined by fundus fluorescein angiography. These results were confirmed via immunohistochemistry using anti-CD31, while a TUNEL assay showed that rAAV2-shmTOR-SD possesses anti-apoptotic properties as well. The qualities exhibited by rAAV2-shmTOR-SD demonstrate its potential as a human gene therapeutic for the treatment of wet AMD.

12.
Genome Res ; 29(6): 978-987, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31123082

RESUMO

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.


Assuntos
Código das Histonas , Histonas/metabolismo , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Peptídeos/síntese química , Peptídeos/metabolismo , Proteômica/métodos , Controle de Qualidade , Reprodutibilidade dos Testes , Fluxo de Trabalho
13.
J Proteome Res ; 18(4): 1893-1901, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30781952

RESUMO

The standard approach for proteomic data acquisition of isobaric-tagged samples by mass spectrometry is data-dependent acquisition. This semistochastic, identification-first paradigm generates a wealth of peptide-level data without regard to relative abundance. We introduce a data acquisition concept called sequential windowed acquisition of reporter masses (SWARM). This approach performs quantitation first, thereby allowing subsequent acquisition decisions to be predicated on user-defined patterns of reporter ion intensities. The efficacy of this approach is validated through experiments with both synthetic mixtures of Escherichia coli ribosomes spiked into human cell lysates at known ratios and the quantitative evaluation of the human proteome's response to the inhibition of cullin-based protein ubiquitination via the small molecule MLN4924. We find that SWARM-informed parallel reaction monitoring acquisitions display effective acquisition biasing toward analytes displaying quantitative characteristics of interest, resulting in an improvement in the detection of differentially abundant analytes. The SWARM concept provides a flexible platform for the further development of new acquisition methods.


Assuntos
Proteoma , Proteômica/métodos , Espectrometria de Massas em Tandem , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Escherichia coli/química , Células HEK293 , Humanos , Peptídeos/análise , Peptídeos/química , Proteoma/análise , Proteoma/química
14.
Genome res, v. 29, n. 6, p. 978-987, jul. 2019
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-2774

RESUMO

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.

15.
Genome Res ; 29: 978-987, 2019.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib17331

RESUMO

DNA and histone proteins define the structure and composition of chromatin. Histone post-translational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitive proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-day sample preparation that includes histone purification, derivatization and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 hours from start to finish. This protocol includes 4 hours of histone extraction, 3 hours of derivatization and digestion, and only 1 minute of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high throughput screening of >1,000 samples per day using a single mass spectrometer

16.
Genome res. ; 29(6): p. 978-987, 2019.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib16051

RESUMO

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.

17.
Invest Ophthalmol Vis Sci ; 59(13): 5398-5407, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30452593

RESUMO

Purpose: With anti-VEGF-based treatments for wet AMD requiring frequent injections, it is often burdensome to both patients and healthcare providers. To explore its possibility as a desirable alternative, we investigated the therapeutic potential of a recombinant adeno-associated virus 2 expressing a soluble variant of VEGF receptor-1 (rAAV2-sVEGFRv-1) in a laser-induced choroidal neovascularization (CNV) model, as CNV is a defining feature of AMD progression. Methods: C57/B6 mice were intravitreally administered with rAAV2-sVEGFRv-1, rAAV2-GFP, or clinically used bevacizumab after CNV lesions were induced via laser photocoagulation. Immunostaining was performed with phalloidin and CD31 to measure CNV extensiveness, F4/80 and CD11b for inflammatory cell infiltration, and pan-cytokeratin to visualize fibrotic progression. Results: rAAV2-sVEGFRv-1 (5.0 × 107 viral genomes) possesses antiangiogenic, anti-inflammatory, and antifibrotic properties. rAAV2-sVEGFRv-1 was demonstrated to significantly decrease retinal CNV lesion size (1336 ± 186) when compared to rAAV2-GFP-treated (2949 ± 437, P = 0.0043), mock-treated (3075 ± 265, P = 0.0013), and bevacizumab-treated models (995 ± 234). Infiltration by inflammatory cells significantly decreased with rAAV2-sVEGFRv-1 administration, while groups treated with rAAV2-GFP did not. Additionally, antiapoptotic activity was observed via TUNEL assay in rAAV2-sVEGFRv-1 (16.0 ± 3.6) and rAAV2-GFP (46.0 ± 7.5, P = 0.003). Overall, the rAAV2-sVEGFRv-1 viral vector was positively comparable to bevacizumab, indicating it as effective as approved therapeutics. Conclusions: The ability of a low dose of rAAV2-sVEGFRv-1 to exert a therapeutically relevant anti-VEGF effect in a CNV model is demonstrated, and strongly suggests gene therapy as an effective and convenient treatment for sustained VEGF suppression.


Assuntos
Neovascularização de Coroide/terapia , Terapia Genética , Parvovirinae/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Western Blotting , Neovascularização de Coroide/diagnóstico , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
18.
Ann Neurol ; 84(5): 766-780, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30295347

RESUMO

OBJECTIVE: Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function. METHODS: Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples. RESULTS: Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology. INTERPRETATION: Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.


Assuntos
Autofagia/genética , Lisossomos/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Canais de Potássio/deficiência , Idade de Início , Pré-Escolar , Feminino , Humanos , Lactente , Lisossomos/patologia , Masculino , Mutação , Linhagem , Canais de Potássio/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Proc Natl Acad Sci U S A ; 115(40): 10022-10027, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224458

RESUMO

All cells obtain 2'-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, ß; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in ß. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the ß is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.


Assuntos
Di-Hidroxifenilalanina/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Radicais Livres/química , Ribonucleotídeo Redutases/química , Tirosina/química , Di-Hidroxifenilalanina/metabolismo , Proteínas de Escherichia coli/metabolismo , Radicais Livres/metabolismo , Ribonucleotídeo Redutases/metabolismo , Tirosina/metabolismo
20.
Mol Ther Methods Clin Dev ; 9: 90-98, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29766021

RESUMO

Adeno-associated virus (AAV) vector is a promising platform technology for ocular gene therapy. Recently clinical successes to treat choroidal neovascularization (CNV) in wet type age-related macular degeneration have been reported. However, because pathologic conditions of the retina may alter the tropism of viral vectors, it is necessary to evaluate the transduction efficiency of different serotypes of AAV vectors in the retinas with CNVs. Here, we show the patterns and efficacy of transduction of AAV2, -5, and -8 vectors in a laser-induced CNV mouse model. C57BL/6J mice were subjected to unilateral laser photocoagulation on the right eye to induce CNV 5 days prior to intravitreal injection of AAV2, -5, and -8 capsids expressing EGFP. Transduction was increased around CNV lesions for all AAV capsid types, and AAV2 resulted in the highest transduction efficiency. In the absence of CNV, the AAV2 vector transduced ganglion and inner nuclear layer (INL) cells, and AAV5 and AAV8 transduced only a small proportion of cells in the retinal ganglion cell layer. CNV increased AAV2 vector expression throughout the retina and in and around CNVs; the transduced cells included retinal ganglion cells, Müller cells, cells from the INL and outer nuclear layer (ONL), photoreceptors, and retinal pigment epithelium (RPE) cells. Inflammatory cells and endothelial cells in CNVs were also transduced by AAV2. AAV5 and AAV8 were transduced in retinal ganglion, Müller, INL, ONL, and RPE cells in a localized pattern, and only endothelial cells at the surface of CNV lesions showed EGFP expression. Taken together, CNV formation resulted in enhanced transduction of AAV2, -5, and -8, and AAV2 exhibited the highest transduction efficiency in cells in CNV lesions.

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